U.S. patent application number 09/886942 was filed with the patent office on 2002-06-27 for novel chimeric promoters.
Invention is credited to Punnonen, Juha, Semyonov, Andrey, Wright, Anne.
Application Number | 20020081708 09/886942 |
Document ID | / |
Family ID | 22796670 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020081708 |
Kind Code |
A1 |
Punnonen, Juha ; et
al. |
June 27, 2002 |
Novel chimeric promoters
Abstract
This invention provides novel chimeric promoter/enhancers. The
chimeric promoter/enhancers are particularly suitable for directing
gene expression in mammalian cells.
Inventors: |
Punnonen, Juha; (Belmont,
CA) ; Wright, Anne; (Woodside, CA) ; Semyonov,
Andrey; (San Francisco, CA) |
Correspondence
Address: |
LAW OFFICES OF JONATHAN ALAN QUINE
P O BOX 458
ALAMEDA
CA
94501
|
Family ID: |
22796670 |
Appl. No.: |
09/886942 |
Filed: |
June 21, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60213829 |
Jun 23, 2000 |
|
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Current U.S.
Class: |
435/235.1 ;
435/5; 536/23.72 |
Current CPC
Class: |
C12N 15/1058 20130101;
G16B 30/00 20190201; C12Q 1/6837 20130101; C12N 15/1034 20130101;
G16B 30/10 20190201; C12N 2800/108 20130101; C12N 15/67 20130101;
C12N 15/1086 20130101; Y02A 50/30 20180101; C12N 15/85
20130101 |
Class at
Publication: |
435/235.1 ;
435/5; 536/23.72 |
International
Class: |
C12Q 001/70; C07H
021/04; C12N 007/01 |
Goverment Interests
[0002] This invention was made in part with government support
under a grant awarded by the Defense Advanced Research Projects
Agency (DARPA) (Grant No. N65236-98-1-5401). The Government may
have certain rights in the invention.
Claims
What is claimed is:
1. An isolated or recombinant nucleic acid comprising a
polynucleotide sequence selected from the group consisting of: (a)
a polynucleotide sequence selected from SEQ ID NO: 1 to SEQ ID
NO:18 or a complementary polynucleotide sequence thereof; (b) a
polynucleotide sequence that has at least about 97% sequence
identity to at least one sequence from the group consisting of SEQ
ID NO: 1 to SEQ ID NO: 18 or a complementary polynucleotide
sequence thereof; (c) a polynucleotide sequence that has at least
about 80% sequence identity to at least one sequence from the group
consisting of SEQ ID NO: 1 to SEQ ID NO: 18, or a complementary
polynucleotide sequence thereof, wherein said polynucleotide
sequence promotes expression of an operably linked transgene at a
level that is greater than the level of expression of the same
transgene when operably linked to a human CMV promoter
polynucleotide sequence; and (d) a polynucleotide sequence
comprising a fragment of (a), (b), or (c), wherein said fragment
promotes expression of an operably linked transgene at a level that
is greater than the level of expression of the same transgene when
operably linked to a human CMV promoter polynucleotide
sequence.
2. The nucleic acid of claim 1, comprising a polynucleotide
sequence of (b), wherein said polynucleotide sequence promotes
expression of an operably linked transgene at a level that is equal
to or greater than the level of expression of the same transgene
when operably linked to a human CMV promoter polynucleotide
sequence.
3. The nucleic acid of claim 1, wherein the human CMV promoter
polynucleotide sequence is a Towne or AD169 human CMV promoter
polynucleotide sequence.
4. The nucleic acid of claim 1, comprising a polynucleotide
sequence selected from the group consisting of SEQ ID NO: 1 to SEQ
ID NO: 18 or a complementary polynucleotide sequence thereof.
5. The nucleic acid of claim 1, comprising a polynucleotide
sequence that has at least about 97% sequence identity to at least
one sequence from the group consisting of SEQ ID NO: 1 to SEQ ID
NO: 18 or a complementary polynucleotide sequence thereof.
6. The nucleic acid of claim 1, comprising a polynucleotide
sequence that has at least about 98% sequence identity to at least
one sequence from the group consisting of SEQ ID NO: 1 to SEQ ID
NO: 18 or a complementary polynucleotide sequence thereof.
7. The nucleic acid of claim 1, comprising a polynucleotide
sequence that has at least about 99% sequence identity to at least
one sequence from the group consisting of SEQ ID NO: 1 to SEQ ID
NO: 18 or a complementary polynucleotide sequence thereof.
8. The nucleic acid of claim 1, comprising a polynucleotide
sequence that has at least about 80% sequence identity to at least
one sequence from the group consisting of SEQ ID NO: 1 to SEQ ID
NO: 18, or a complementary polynucleotide sequence thereof, wherein
said polynucleotide sequence promotes expression of an operably
linked transgene at a level that is greater than the level of
expression of the same transgene when operably linked to a human
CMV promoter polynucleotide sequence.
9. The nucleic acid of claim 1, comprising a polynucleotide
sequence comprising a fragment of claim 1 (a), (b), or (c), wherein
said fragment promotes expression of an operably linked transgene
at a level that is greater than the level of expression of the same
transgene when operably linked to a human CMV promoter
polynucleotide sequence.
10. An isolated or recombinant nucleic acid comprising a fragment
of one sequence from the group consisting of SEQ ID NO: 1 to SEQ ID
NO: 18 or a fragment of a complementary polynucleotide sequence
thereof, wherein the fragment comprises a unique subsequence.
11. The nucleic acid of claim 10, wherein the fragment promotes the
expression of a transgene to which the fragment is operably
linked.
12. An isolated or recombinant nucleic acid comprising a
polynucleotide sequence that hybridizes under highly stringent
conditions over substantially the entire length of a polynucleotide
sequence of claim 1 (a), (b), (c), or (d).
13. The nucleic acid of claim 12, wherein the highly stringent
conditions are selected such that a polynucleotide sequence
selected from SEQ ID NO: 1 to SEQ ID NO: 18 hybridizes to its
perfect complement with at least a 5-fold higher signal to noise
ratio than for hybridization of the perfect complement to a control
nucleic acid comprising a human CMV promoter polynucleotide
sequence.
14. The nucleic acid of claim 1, comprising a polynucleotide
sequence that promotes the expression of an operably linked
transgene at a level that differs from the expression level of the
same transgene when operably linked to a nucleic acid sequence
corresponding to a human CMV promoter polynucleotide sequence.
15. The nucleic acid of claim 14, wherein the transgene is
luciferin luciferase, and transgene expression level is determined
in an in vitro luciferase assay.
16. The nucleic acid of claim 14, wherein the transgene is
.beta.-galactosidase, the transgene is expressed in vivo, and
transgene expression level is determined by measuring the serum
titer of anti-.beta.-galactosidase antibodies.
17. The nucleic acid of claim 14, wherein the polynucleotide
sequence promotes the expression of an operably linked transgene at
a level that is higher than the highest expression level of the
same transgene when operably linked to a nucleic acid sequence
corresponding to a human CMV promoter polynucleotide sequence.
18. The nucleic acid of claim 17, wherein polynucleotide sequence
promotes the expression of an operably linked transgene at a level
that is 2-fold higher than the highest expression level of the same
transgene when operably linked to a nucleic acid sequence
corresponding to a human CMV promoter polynucleotide sequence.
19. The nucleic acid of claim 14, wherein the polynucleotide
sequence promotes the expression of an operably linked transgene at
a level that is lower than the lowest expression level of the same
transgene when operably linked to a nucleic acid sequence
corresponding to a human CMV promoter polynucleotide sequence.
20. The nucleic acid of claim 19, wherein polynucleotide sequence
promotes the expression of an operably linked transgene at a level
that is 2-fold lower than the lowest expression level of the same
transgene when operably linked to a nucleic acid sequence
corresponding to a human CMV promoter polynucleotide sequence.
21. The nucleic acid of claim 1, wherein the nucleic acid comprises
a deletion of one or more nucleotides in a region corresponding to
about nucleotides 830-835 or 841-844 of the consensus sequence
shown in FIG. 8.
22. The nucleic acid of claim 21, wherein the nucleic acid
comprises a deletion of nucleotides corresponding to about
nucleotides 830-835 or 841-844 of the consensus sequence.
23. The nucleic acid of claim 22, wherein the nucleic acid
comprises a deletion of nucleotides corresponding to about
nucleotides 830-835 and 841-844 of the consensus sequence.
24. The nucleic acid of claim 1, wherein the nucleic acid comprises
a Rhesus monkey CMV promoter polynucleotide sequence at about
nucleotide positions 817-863, numbered according to the consensus
sequence shown in FIG. 8.
25. The nucleic acid of claim 1, wherein the nucleic acid comprises
a polynucleotide sequence selected from GACGCCGGAGG and
GACGTCGGAG.
26. The nucleic acid of claim 1, wherein the nucleic acid comprises
an insertion of a nucleotide, as compared to the human Towne CMV
promoter sequence, after nucleotide position 853, numbered
according to the consensus sequence shown in FIG. 8.
27. The nucleic acid of claim 1, wherein the nucleic acid comprises
a deletion of one or more nucleotides in a region corresponding to
about nucleotides 684-735 of the consensus sequence shown in FIG.
8.
28. The nucleic acid of claim 27, wherein the nucleic acid
comprises a deletion of any nucleotides corresponding to about
nucleotides 684-735 of the consensus sequence.
29. The nucleic acid of claim 1, wherein the nucleic acid comprises
the polynucleotide sequence AATGGGCGGTC.
30. The nucleic acid of claim 1, wherein the nucleic acid does not
comprise CMV promoter nucleic acid residues beyond about nucleotide
residue 909, numbered according to the consensus sequence shown in
FIG. 8.
31. The nucleic acid of claim 1, wherein the nucleic acid comprises
a polynucleotide sequence comprising nucleic acid residue 1 to
about nucleotide residue 930, numbered according to the consensus
sequence shown in FIG. 8.
32. The nucleic acid of claim 31, wherein the nucleic acid does not
comprise CMV promoter nucleic acid residues beyond about nucleotide
residue 930, numbered according to the consensus sequence.
33. The nucleic acid of claim 1, wherein the nucleic acid comprises
a polynucleotide sequence comprising nucleic acid residue 1 to
nucleotide residue 932, numbered according to the consensus
sequence shown in FIG. 8.
34. The nucleic acid of claim 33, wherein the nucleic acid does not
comprise CMV nucleotide residues beyond nucleotide residue 932,
numbered according to the consensus sequence shown in FIG. 8.
35. The nucleic acid of claim 1, wherein the nucleic acid comprises
a deletion of one or more nucleotides in a region corresponding to
about nucleotide residues 319-512 of the consensus sequence shown
in FIG. 8.
36. The nucleic acid of claim 35, wherein the nucleic acid
comprises a deletion of nucleotides corresponding to about
nucleotide residues 319-512 of the consensus sequence.
37. The nucleic acid of claim 1, wherein the polynucleotide
sequence comprises SEQ ID NO:21 or a complementary polynucleotide
sequence thereof.
38. The nucleic acid of claim 1, wherein the polynucleotide
sequence comprises SEQ ID NO:8 (6A8) or a complementary
polynucleotide sequence thereof.
39. The nucleic acid of claim 1, wherein the polynucleotide
sequence comprises SEQ ID NO: 11 (6F6) or a complementary
polynucleotide sequence thereof.
40. The nucleic acid of claim 1, wherein the polynucleotide
sequence comprises SEQ ID NO:6 (3C9) or a complementary
polynucleotide sequence thereof.
41. The nucleic acid of claim 1, wherein the polynucleotide
sequence comprises SEQ ID NO:9 (6B2) or a complementary
polynucleotide sequence thereof.
42. The nucleic acid of claim 1, wherein the polynucleotide
sequence comprises SEQ ID NO:2 (11E2) or a complementary
polynucleotide sequence thereof.
43. The nucleic acid of claim 1, wherein the polynucleotide
sequence comprises SEQ ID NO:3 (12C9) or a complementary
polynucleotide sequence thereof.
44. The nucleic acid of claims 1, 10 or 12, wherein the
polynucleotide sequence is operably linked to a transgene to form
an expression cassette.
45. The nucleic acid of claim 44, wherein the transgene is a viral
gene.
46. The nucleic acid of claim 44, wherein the transgene encodes a
polypeptide selected from the group consisting of an immunogen, an
immunomodulatory molecule, an antigen, an adjuvant, an allergen, an
antibody, a bacterial toxin, a cytokine, a cytokine receptor, and a
co-stimulatory molecule.
47. The nucleic acid of claim 46, wherein the transgene encodes an
antigen selected from the group consisting of a cancer antigen, a
hepatitis B surface antigen, a hepatitis A antigen, and a hepatitis
C antigen.
48. The nucleic acid of claim 46, wherein the transgene encodes a
co-stimulatory molecule comprising a polypeptide that binds to a
CD28 or CTLA-4 receptor.
49. A composition produced by the cleaving of one or more nucleic
acids of claims 1, 10, or 12, wherein the cleaving comprises
mechanical, chemical, or enzymatic cleavage.
50. The composition of claim 49, wherein the cleaving comprises
enzymatic cleavage with a restriction endonuclease, an RNAse or a
DNAse.
51. A composition produced by a process comprising incubating one
or more nucleic acids of claims 1, 10, or 12 in the presence of
deoxyribonucleotide triphosphates and a nucleic acid
polymerase.
52. The composition of claim 51, wherein the nucleic acid
polymerase is a thermostable polymerase.
53. A method of producing a modified or recombinant nucleic acid
comprising mutating or recombining a nucleic acid of claims 1, 10,
or 12.
54. The method of claim 53, comprising recursively recombining the
nucleic acid with one or more additional nucleic acids.
55. The method of claim 54, wherein the one or more additional
nucleic acids promote the expression of an operably linked
transgene.
56. The method of claim 54, wherein the recursive recombination is
performed in vitro.
57. The method of claim 54, wherein the recursive recombination is
performed in vivo.
58. The method of claim 54, wherein the recursive recombination
produces at least one library of recombinant nucleic acids, which
library comprises at least one recombinant nucleic acid that
promotes the expression of an operably linked transgene.
59. The method of claim 53 additionally comprising assaying the
modified or recombinant nucleic acid produced by the method for the
ability to promote the expression of an operably linked
transgene.
60. A nucleic acid library produced by the method of claim 53.
61. A nucleic acid library comprising two or more nucleic acids of
claims 1, 10, or 12.
62. A vector comprising at least one nucleic acid of claims 1, 10,
12 or 44.
63. The vector of claim 62, wherein the vector is an expression
vector.
64. The vector of claim 62, wherein the vector is selected from a
plasmid, a cosmid, a phage, a virus or fragment thereof, a
bacterial artificial chromosome (BAC), a yeast artificial
chromosome (YAC).
65. A cell comprising the nucleic acid of claims 1, 10, or 12 or
the vector of claim 62.
66. The cell of claim 65, wherein the cell comprises a human
cell.
67. A population of cells comprising the library of claims 60 or
61.
68. A composition comprising the nucleic acid of claims 1, 10, or
12 or the vector of claim 62 and a carrier.
69. The composition of claim 68, wherein the excipient is a
pharmaceutically acceptable carrier.
70. The composition of claim 48, wherein the nucleic acid or vector
is present in the composition in an amount sufficient to introduce
the nucleic acid or vector into cells of a subject, when the
composition is administered to the subject.
71. A composition comprising the nucleic acid of claims 1, 10, or
12 or the vector of claim 62 in an amount sufficient to introduce
the nucleic acid or vector into cells of a subject, when the
composition is administered to the subject.
72. The composition of claims 70 or 71, wherein the amount is
sufficient to introduce the nucleic acid or vector into cells of a
subject, when the composition is administered to the subject by a
route selected from the group consisting of topical administration,
injection, implantation, oral administration, buccal, vaginal
administration, rectal administration, and inhalation.
73. The composition of claim 75, wherein the composition is
administered to the subject by a route selected from the group
consisting of intradermal, subdermal, subcutaneous, intramuscular,
intravenous, intraperitoneal, and intrathecal.
74. A method of producing a polypeptide, the method comprising: (a)
providing a population of cells comprising a nucleic acid of claims
1, 10, or 12 operably linked to a transgene encoding a polypeptide;
and (b) expressing the polypeptide in at least the subset of the
population of cells or progeny thereof.
75. The method of claim 74, wherein the population of cells is
provided by introducing the nucleic acid operably linked to the
transgene into the population of cells.
76. The method of claim 74, further comprising isolating the
polypeptide from the cells.
77. The method of claim 74, wherein the cells are in culture.
78. The method of claim 77, comprising expressing the polypeptide
by culturing the population or subset of the population of cells or
progeny thereof in a nutrient medium under conditions in which the
nucleic acid promotes expression of the polypeptide.
79. The method of claim 78, further comprising isolating or
recovering the polypeptide from the cells or from the nutrient
medium.
80. The method of claim 74, wherein the cells comprise mammalian
cells selected from fertilized oocytes, embryonic stem cells, or
pluripotent stem cells, the method further comprising generating a
transgenic mammal expressing the polypeptide.
81. The method of claim 80, further comprising recovering the
polypeptide from the transgenic mammal or a byproduct of the
transgenic mammal.
82. The method of claim 74, wherein the cells are in vivo in a
subject.
83. The method of claim 82, wherein the nucleic acid is introduced
into cells in culture, and the cells are subsequently introduced
into the subject.
84. The method of claim 82, wherein the nucleic acid is introduced
into the cells of the subject by administering the nucleic acid
directly to the subject.
85. The method of claim 84, wherein the nucleic acid is
administered to the subject by a route selected from the group
consisting of topical administration, injection, implantation, oral
administration, vaginal administration, rectal administration, and
inhalation.
86. The method of claim 85, wherein the nucleic acid is
administered to the subject by a route selected from the group
consisting of intradermal, subdermal, subcutaneous, intramuscular,
intravenous, intraperitoneal, and intrathecal.
87. The method of claim 84, wherein the nucleic acid is
administered to the subject by topical administration, injection,
or using a gene gun.
88. The method of claim 82, wherein the subject is a human.
89. The method of claim 82, wherein the polypeptide is expressed in
an amount sufficient to produce a desired effect in the
subject.
90. The method of claim 89, wherein the desired effect comprises an
immunogenic effect, a prophylactic effect, or a therapeutic
effect.
91. A nucleic acid of claims 1, 10, or 12 for use in producing an
immunogenic effect, a prophylactic effect, or a therapeutic effect
in a subject.
92. The nucleic acid of claim 91, wherein the subject is a
human.
93. A kit comprising a nucleic acid of claims 1, 10, 12, or 44.
94. A kit comprising a vector of claims 62 or 63.
95. A database comprising one or more character strings
corresponding to a polynucleotide sequence selected from SEQ ID NO:
1 to SEQ ID NO: 18 or a complementary polynucleotide sequence
thereof.
96. A database comprising one or more character strings
corresponding to a unique subsequence of a polynucleotide sequence
selected from SEQ ID NO: 1 to SEQ ID NO: 18 or a uniques
subsequence of a complementary polynucleotide sequence thereof.
97. The database of claims 95 or 96, wherein the one or more
character strings is recorded in a computer-readable medium.
98. A method for manipulating a sequence record in a computer
system, the method comprising: (a) reading a character string
corresponding to a polynucleotide sequence selected from SEQ ID NO:
1 to SEQ ID NO: 18, or a complementary polynucleotide sequence
thereof; (b) performing an operation on the character string; and
(c) returning a result of the operation.
99. A method for manipulating a sequence record in a computer
system, the method comprising: (a) reading a character string
corresponding to a unique subsequence of a polynucleotide sequence
selected from SEQ ID NO:1 to SEQ ID NO:18 or a uniques subsequence
of a complementary polynucleotide sequence thereof; (b) performing
an operation on the character string; and (c) returning a result of
the operation.
100. The method of claims 98 or 99, wherein the user selects the
character string from a database or inputs the character string
into the computer system.
101. The method of claims 98 or 99, comprising performing one or
more operations selected from among: a local sequence comparison, a
sequence alignment, a sequence identity or similarity search, a
sequence identity or similarity determination, a nucleic acid motif
determination, a hypothetical translation, a determination of a
restriction map, a sequence recombination, or a BLAST
determination.
102. The method of claim 101, comprising aligning the selected
character string with one or more additional character strings
corresponding to a polynucleotide sequence.
103. The method of claim 101, wherein the operation comprises
transmitting the character string to a device capable of producing
a nucleic acid comprising the polynucleotide sequence corresponding
to the character string.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to and benefit of U.S.
Provisional Patent Application Serial No. 60/213,829, filed on Jun.
23, 2000, the full disclosure of which is incorporated herein by
reference in its entirety for all purposes.
COPYRIGHT NOTIFICATION
[0003] Pursuant to 37 C.F.R. 1.71(e), Applicants note that a
portion of this disclosure contains material which is subject to
copyright protection. The copyright owner has no objection to the
facsimile reproduction by anyone of the patent document or patent
disclosure, as it appears in the Patent and Trademark Office patent
file or records, but otherwise reserves all copyright rights
whatsoever.
FIELD OF THE INVENTION
[0004] This invention pertains to the field of transcriptional
promoters and enhancers for use in expressing genes in cells.
BACKGROUND OF THE INVENTION
[0005] A key to many aspects of genetic engineering is the ability
to obtain a sufficient level of expression of a gene of interest.
The use of genetic engineering to produce proteins of commercial
importance, such as erythropoietin, tissue plasminogen activator,
and many others, is well established. However, the cost of
producing such products could be decreased by the ability to
express a gene that encodes the protein at a higher level. Gene
therapy, which involves the introduction of a nucleic acid into
cells of a patient to express the nucleic acid for some therapeutic
purpose, also depends upon obtaining a sufficient level of
expression to achieve the desired result. In other applications,
delivery of genes encoding a toxin (e.g., diphtheria toxin, ricin,
tk) can be used to kill cancer cells, and other genes can be
specifically tailored to kill infectious organisms. Again,
obtaining an optimized or sufficient level of expression is a key
to success. Genetic vaccines, which express proteins that can
induce and/or modulate an immune response, also require adequate
levels of gene expression.
[0006] Therefore, a need exists for promoters and enhancers that
can provide appropriate levels of gene expression (e.g., great,
intermediate, or low gene expression levels), as needed for the
particular application or purpose, in target cells of interest. The
present invention fulfills this and other needs.
SUMMARY OF THE INVENTION
[0007] The invention provides novel chimeric or recombinant
promoter/enhancers for use in expressing genes in mammalian and
other cells. The promoters were obtained by performing DNA
shuffling on several isolates of the cytomegalovirus (CMV)
immediate early (IE) promoter. The resulting chimeric
promoter/enhancers were subjected to screening to identify those
that exhibit improved expression, in vitro, as well as in mammals
in vivo.
[0008] Accordingly, one aspect of the invention is an isolated or
recombinant nucleic acid comprising a polynucleotide sequence
selected from:
[0009] (a) a polynucleotide sequence selected from SEQ ID NO: 1 to
SEQ ID NO: 18 or a complementary polynucleotide sequence
thereof;
[0010] (b) a polynucleotide sequence that has at least about 97%
sequence identity to at least one sequence selected from SEQ ID NO:
1 to SEQ ID NO: 18 or a complementary polynucleotide sequence
thereof;
[0011] (c) a polynucleotide sequence that has at least about 80%
sequence identity to at least one sequence from the group
consisting of SEQ ID NO: 1 to SEQ ID NO: 18, or a complementary
polynucleotide sequence thereof, wherein the polynucleotide
sequence promotes expression of an operably linked transgene at a
level that is greater than the level of expression of the same
transgene when operably linked to a human CMV promoter
polynucleotide sequence; and
[0012] (d) a polynucleotide sequence comprising a fragment of (a),
(b), or (c), wherein the fragment promotes expression of an
operably linked transgene at a level that is greater than the level
of expression of the same transgene when operably linked to a human
CMV promoter polynucleotide sequence. The invention also includes
an isolated or recombinant nucleic acid comprising a polynucleotide
sequence that hybridizes under highly stringent conditions over
substantially the entire length of a polynucleotide sequence of
claim 1 (a), (b), (c), or (d).
[0013] In another embodiment, the invention provides a
polynucleotide sequence comprising a fragment of (a), (b), or (c),
wherein the fragment promotes expression of an operably linked
transgene at a level that is greater than the level of expression
of the same transgene when operably linked to a human CMV promoter
polynucleotide sequence.
[0014] The invention also provides an isolated or recombinant
nucleic acid comprising a fragment of one sequence selected from
SEQ ID NO: 1 to SEQ ID NO: 18 or a fragment of a complementary
polynucleotide sequence thereof, wherein the fragment comprises a
unique subsequence.
[0015] Another aspect of the invention is a composition produced by
the cleaving of on or more nucleic acids of the invention, wherein
the cleaving comprises mechanical, chemical, or enzymatic cleavage.
Also included in the invention is a composition produced by a
incubating one or more nucleic acids of the invention in the
presence of deoxyribonucleotide triphosphates and a nucleic acid
polymerase.
[0016] Other aspects of the invention relate to a method of
producing a modified or recombinant nucleic acid comprising
mutating or recombining a nucleic acid of the invention.
Accordingly, the invention also includes a nucleic acid library
produced by this method, and a nucleic acid library comprising two
or more nucleic acids of the invention.
[0017] In addition, the invention provides a vector comprising at
least one nucleic acid of the invention, a cell comprising a
nucleic acid or vector of the invention, and a population of cells
comprising a library of the invention.
[0018] In another aspect, the invention includes composition
comprising a nucleic acid or vector of the invention and a carrier.
In a preferred variation of this embodiment, the nucleic acid or
vector is present in the composition in an amount sufficient to
introduce the nucleic acid or vector into cells of a subject, when
the composition is administered to the subject.
[0019] The invention also provides a method of producing a
polypeptide, which entails:
[0020] (a) providing a population of cells comprising a nucleic
acid of the invention operably linked to a transgene encoding a
polypeptide; and
[0021] (b) expressing the polypeptide in at least the subset of the
population of cells or progeny thereof.
[0022] The method can, optionally, comprise isolating the
polypeptide from the cells. In a variation of this embodiment, the
method includes introducing the nucleic acid operably linked to the
transgene into the population of cells. The cells can be in culture
or in vivo in a subject. For in vivo applications, the nucleic acid
can be introduced into cells in culture, and the cells can
subsequently be introduced into the subject. Alternatively, the
nucleic acid can be introduced into the cells of the subject by
administering the nucleic acid directly to the subject. In
preferred in embodiments, where the polypeptide is expressed in
vivo, the polypeptide is expressed in an amount sufficient to
produce a desired effect in the subject, such as an immunogenic
effect, a prophylactic effect, or a therapeutic effect.
Accordingly, the invention also includes a nucleic acid of the
invention for use in producing an immunogenic effect, a
prophylactic effect, or a therapeutic effect in a subject.
[0023] In other aspects, the invention provides a kit comprising a
nucleic acid or vector of the invention.
[0024] The invention also encompasses computer-related uses of the
nucleotide sequences of the invention. Thus, the invention provides
a database comprising one or more character strings corresponding
to a polynucleotide sequence selected from SEQ ID NO: 1 to SEQ ID
NO:18 or a complementary polynucleotide sequence thereof and a
database comprising one or more character strings corresponding to
a unique subsequence of a polynucleotide sequence selected from SEQ
ID NO:1 to SEQ ID NO:18 or a unique subsequence of a complementary
polynucleotide sequence thereof.
[0025] The invention also provides a method for manipulating a
sequence record in a computer system, the method comprising:
[0026] (a) reading a character string corresponding to a
polynucleotide sequence selected from SEQ ID NO:1 to SEQ ID NO:18,
or a complementary polynucleotide sequence thereof;
[0027] (b) performing an operation on the character string; and
[0028] (c) returning a result of the operation.
[0029] In another embodiment, the invention provides method for
manipulating a sequence record in a computer system, the method
comprising:
[0030] (a) reading a character string corresponding to a unique
subsequence of a polynucleotide sequence selected from SEQ ID NO:1
to SEQ ID NO:18 or a unique subsequence of a complementary
polynucleotide sequence thereof;
[0031] (b) performing an operation on the character string; and
[0032] (c) returning a result of the operation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 shows a protocol for screening libraries of chimeric
promoter sequences that were produced by shuffling of CMV promoter
sequences ("promoters"). A three-tiered approach to screening such
shuffled chimeric promoter libraries was applied; first, the
library was enriched for good promoter sequences by FACS
(Fluorescence-Activated Cell Sorting) sorting. The best sequences
were then identified by high throughput transfection and FACS
analysis of individual clones. These were subcloned in DNA vaccine
vectors encoding luciferase or .beta.-galactosidase to test
transgene expression and induction of antibody (Ab) responses in
vivo.
[0034] FIG. 2 shows that FACS sorting resulted in enrichment of the
chimeric promoter libraries for chimeric promoters that provide a
greater amount of reporter gene expression. Individual clones from
the round 1 shuffled chimeric promoter library and the enriched
library were assayed by transfection and FACS analysis. This
analysis revealed a higher frequency of strongly expressing clones
in the enriched library.
[0035] FIG. 3 shows that diverse activities of chimeric promoter
sequences are obtained in transfected cells. Transfection and FACS
analysis of individual clones revealed a large diversity of
promoter activities in the chimeric promoter libraries. Results for
vector control and parental clones are presented in lightly colored
bars, dark bars represent shuffled clones. Results are expressed as
mean.+-.SD for 4 independent transfections.
[0036] FIG. 4 shows the amount of luciferase expression obtained in
muscle 7 days after injection of a plasmid expression vector that
comprised a luciferase gene under the control of a shuffled versus
a control CMV promoter. Mice were injected with 10 .mu.g plasmid in
each tibialis anterior (TA) muscle; muscles were collected at 7
days post-injection, homogenized, and the luciferase content
assayed. Results are expressed as mean.+-.SEM for 32 samples.
[0037] FIG. 5 shows a comparison of luciferase expression from a
plasmid vector injected intramuscularly comprising a luciferase
gene and a promoter sequence corresponding to clone 6A8 or a
parental clone, where the luciferase gene was under the control of
the promoter. Shuffled clone 6A8 gave 2-fold higher luciferase
expression than did AD169 and Towne parental clones (p<0.05,
t-test).Results are expressed as mean.+-.SEM for 32 samples.
[0038] FIG. 6A shows the antibody titer obtained following
injection of mice with .beta.-galactosidase-encoding plasmids. Mice
were injected with 10.mu.g plasmid on days 0 and 15; serum was
collected on days 14 and 28 to measure antibody levels by ELISA.
Shuffled clone 6B2 gave the highest antibody responses at day 28
post-injection. Results are expressed as mean.+-.SEM for 8-20
samples.
[0039] FIG. 6B shows the antibody titer obtained in a similar study
in which mice were injected with 4.mu.g plasmid.
[0040] FIG. 7 shows that the chimeric promoter 6A8 is functional in
human muscle tissue. Luciferase was measured in homogenates of
human fetal muscle 2 days after injection of luciferase-encoding
plasmids. Results are expressed as mean.+-.SEM for 3-6 injections
for each clone.
[0041] FIGS. 8A-8I shows an alignment of the polynucleotide
sequences of WT human AD169 and Towne CMV promoters (SEQ ID NOS: 19
and 20) and exemplary polynucleotide sequences of the invention
(SEQ ID NOS: 1-18). The arrow located between the nucleic acid
residue positions equivalent to nucleic acid residues 808-809 of
the human Towne CMV promoter sequence indicates the transcription
start site. The predicted boundary between the first exon and the
first intron is also indicated by an arrow between nucleic acid
residues 930 and 931 of the human Towne CMV promoter sequence. The
last sequence shown in the alignment (SEQ ID NO:21) represents a
"consensus sequence" of aligned polynucleotide sequences. The
alignment was prepared using the CLUSTALW multiple sequence
alignment algorithm, a part of the Vector NTI version 6 sequence
analysis software package (Informax, Bethesda, Md.). The CLUSTALW
program initially performs multiple pairwise comparisons between
groups of sequences and then assembles the pairwise alignments into
a multiple alignment based on homology. For the initial pairwise
alignments, Gap Open and Gap Extension penalties were 10 and 0.1,
respectively. For the multiple alignments, Gap Open penalty was 10,
and the Gap Extension penalty was 0.05. The protein weight matrix
employed was the BLOSUM62 matrix.
[0042] FIG. 9 shows an example of a vector that is useful for
screening to identify improved promoters from a library of shuffled
promoter nucleic acids. Shuffled putative promoters are inserted
into the vector upstream of a reporter gene for which expression is
readily detected. For many applications, it is desirable that the
product of the reporter gene be a cell surface protein so that
cells which express high levels of the reporter gene can be sorted
using flow cytometry-based cell sorting using the reporter gene
product. Examples of suitable reporter genes include, for example,
luciferase, .beta.-galactosidase, or mAb179 epitopes. A
polyadenylation region is typically placed downstream of the
reporter gene (SV40 polyA is illustrated). The vector can also
include a second reporter gene an internal control (GFP; "green
fluorescent protein"); this gene is linked to a promoter
(SR.alpha.p) described herein. The vector also typically includes a
selectable marker (kanamycin/neomycin resistance is shown), and
origins of replication that are functional in mammalian (SV40 ori)
and/or bacterial (pUC ori) cells.
[0043] FIGS. 10A-10D shows an alignment of the polynucleotide
sequences of WT of the promoter/enhancer regions of the WT Rhesus
monkey (SEQ ID NO: 22), Vervet monkey (SEQ ID NO:23), and human
Towne (SEQ ID NO:20) CMV isolates.
DETAILED DESCRIPTION
[0044] Definitions
[0045] The term "gene" broadly refers to any segment of DNA
associated with a biological function. Genes include coding
sequences and/or regulatory sequences required for their
expression. Genes also include non-expressed DNA nucleic acid
segments that, e.g., form recognition sequences for other proteins
(e.g., promoter, enhancer, or other regulatory regions). Genes can
be obtained from a variety of sources, including cloning from a
source of interest or synthesizing from known or predicted sequence
information, and may include sequences designed to have desired
parameters.
[0046] A "promoter," as used herein, is a DNA regulatory region
that is capable of binding RNA polymerase in a cell (or in vitro
transcription system) and initiating transcription of a downstream
(3' direction) coding sequence. Often, a promoter is associated
with one or more "enhancers" which can provide further regulation
of transcription. Enhancers can also be found upstream of the
promoter, as well as downstream. A promoter is sometimes bounded at
its 3' terminus by the transcription initiation site, but often the
promoter/enhancer region includes additional sequences that affect
transcription and are found downstream of the transcription
initiation site. A promoter extends upstream (5' direction) from
the transcription initiation site to include the minimum number of
bases or elements necessary to initiate transcription at levels
detectable above background. The entire promoter/enhancer region
can extend farther upstream to include additional sequences that
affect gene expression. Within the promoter/enhancer sequences will
be found a transcription initiation site (conveniently defined for
example, by mapping with nuclease S1), as well as protein binding
domains (consensus sequences) responsible for the binding of RNA
polymerase, transcription factors, and other molecules that are
involved in transcription. Eukaryotic class II promoters will
often, but not always, contain "TATA" boxes and "CAAT" boxes. The
human cytomegalovirus (hCMV) immediate early promoter/enhancer (the
"CMV promoter," as used herein), for example, also includes, for
example, repeat elements of 19, 18 and 21 base pairs (bp) that
include binding sites for CREB/ATF, NF-.cndot. B/rel, SP-1 and YY-1
binding sites, respectively (Stinski, MF (1999), in Gene Expression
Systems: Using Nature for the Art of Expression, Academic Press,
pp. 211-233).
[0047] A "chimeric promoter/enhancer" is a non-naturally occurring
promoter/enhancer that includes nucleotides from more than one
source nucleic acid. The source nucleic acids can be naturally
occurring nucleic acids (e.g., nucleic acids from different
isolates or species used in family shuffling), but also can be
non-naturally occurring nucleic acids. Those of skill in the art
will appreciate that the phrase "nucleotides from more than one
source nucleic acid" describes the identity of a particular residue
at a particular position in a chimeric nucleic acid or the sequence
of nucleotides in a particular region of the chimeric nucleic acid.
Thus, two polynucleotide sequences in a chimeric nucleic acid are
said to be from different source nucleic acids if the
polynucleotide sequences are each identical to a polynucleotide
sequence in one of the source nucleic acids. This language does not
imply that the chimeric nucleic acid was necessarily formed by
joining polynucleotide sequences obtained directly from the source
nucleic acids, although the invention encompasses chimeric nucleic
acids formed in this mannter. As used herein, the term
"promoter/enhancer" can refer to either a promoter sequence, as
defined above, or an enhancer sequence, or a polynucleotide
sequence including both types of sequences.
[0048] "Nucleic acid derived from a gene" refers to a nucleic acid
for whose synthesis the gene, or a subsequence thereof, has
ultimately served as a template. Thus, an mRNA, a cDNA reverse
transcribed from an MRNA, an RNA transcribed from that cDNA, a DNA
amplified from the cDNA, an RNA transcribed from the amplified DNA,
etc., are all derived from the gene and detection of such derived
products is indicative of the presence and/or abundance of the
original gene and/or gene transcript in a sample.
[0049] The term "nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form. Unless specifically limited, the term
encompasses nucleic acids containing known analogues of natural
nucleotides which have similar function and are metabolized in a
manner similar to naturally occurring nucleotides. The term
"nucleic acid" is used interchangeably with the term
"polynucleotide" and encompasses genes, cDNA, and MRNA encoded by a
gene.
[0050] The term "polynucleotide sequence" is a nucleic acid which
comprises a polymer of nucleic acid residues or nucleotides
(A,C,T,U,G, etc. or naturally occurring or artificial nucleotide
analogues), or a character string representing a nucleic acid,
depending on context. Either the given nucleic acid or the
complementary nucleic acid can be determined from any specified
polynucleotide sequence.
[0051] As used herein, the term "complementary" refers to the
capacity for precise pairing between two nucleotides. Thus, if a
nucleotide at a given position of a nucleic acid molecule is
capable of hydrogen bonding with a nucleotide of another nucleic
acid molecule, then the two nucleic acid molecules are considered
to be complementary to one another at that position. The term
"substantially complementary" describes sequences that are
sufficiently complementary to one another to allow for specific
hybridization under stringent hybridization conditions. The term
"perfectly complementary" refers to sequences in which there are no
mismatched nucleotides (i.e., each nucleotide in both sequences can
hydrogen bond with a complementary nucleotide in the other
sequence). One such sequence is said to be the "perfect complement"
of the other.
[0052] Nucleic acids according to the subject invention need not be
identical, but can be substantially identical (or substantially
similar), to the corresponding sequences of the exemplary chimeric
promoter/enhancers described herein. In particular, these nucleic
acids can be modified in a number of ways, including mutation or
recombination, using standard techniques. A variety of diversity
generating protocols are available and described in the art. The
procedures can be used separately, and/or in combination to produce
one or more variants of a nucleic acid or set of nucleic acids, as
well variants of encoded proteins. Individually and collectively,
these procedures provide robust, widely applicable ways of
generating diversified nucleic acids and sets of nucleic acids
(including, e.g., nucleic acid libraries) useful, e.g., for the
engineering or rapid evolution of nucleic acids, proteins,
pathways, cells and/or organisms with new and/or improved
characteristics.
[0053] A "library" of nucleic acids includes at least 2 different
nucleic acids, and preferably at least about 5, 10, 50, 10.sup.2,
10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7 or more different
nucleic acids.
[0054] Variants of the exemplary nucleic acids described herein
generally comprise a sequence substantially similar or
substantially identical (as defined below) to at least one of SEQ
ID NOS:1-18 or a complementary polynucleotide sequence or fragment
thereof.
[0055] The term "sequence identity" means that two polynucleotide
sequences are identical (i.e., on a nucleotide-by-nucleotide basis)
over a window of comparison. The term "percentage of sequence
identity" or "percentage of sequence similarity" is calculated by
comparing two optimally aligned sequences over the window of
comparison, determining the number of positions at which the
identical residues occur in both nucleotide sequences to yield the
number of matched positions, dividing the number of matched
positions by the total number of positions in the window of
comparison (i.e., the window size), and multiplying the result by
100 to yield the percentage of sequence identity (or percentage of
sequence similarity).
[0056] As applied in the context of two nucleic acids, the term
substantial identity or substantial similarity means that the two
nucleic acid sequences, when optimally aligned, such as by the
programs BLAST, GAP or BESTFIT using default gap weights (described
in detail below) or by visual inspection, share at least about 70
percent, 75 percent, 80 percent, 85 percent or 88 percent sequence
identity or sequence similarity, preferably at least about 90
percent, 91 percent, 92 percent, 93 percent or 94 percent sequence
identity or sequence similarity, more preferably at least about 95
percent sequence identity or sequence similarity, or more
(including, e.g., about 96, 97, 98, 98.5, 99, 99.5 or more percent
nucleotide sequence identity or sequence similarity). Preferably,
the substantial identity exists over a region of the sequences that
is at least about 50 residues in length, more preferably over a
region of at least about 100 residues, and most preferably the
sequences are substantially identical over at least about 150
residues or more.
[0057] In one aspect, the present invention provides chimeric CMV
promoter/enhancer homologue nucleic acids having at least about 70,
75, 80, 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 98.5, 99, 99.5,
or more percent sequence identity or sequence similarity with the
nucleic acid sequences of any of SEQ ID NOS:1-18 or complementary
polynucleotide sequences or fragments thereof.
[0058] A preferred example of an algorithm that is suitable for
determining percent sequence identity or sequence similarity is the
FASTA algorithm, which is described in Pearson, W. R. & Lipman,
D. J., (1988) Proc Natl Acad Sci USA 85:2444. See also, W. R.
Pearson, (1996) Methods Enzymology 266:227-258. Preferred
parameters used in a FASTA alignment of DNA sequences to calculate
percent identity or percent similarity are optimized, BL50 Matrix
15: -5, k-tuple=2; joining penalty=40, optimization=28; gap penalty
-12, gap length penalty=-2; and width=16.
[0059] Other preferred examples of algorithms that are suitable for
determining percent sequence identity or sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al., (1977) Nuc Acids Res 25:3389-3402 and Altschul et al.,
(1990) J Mol Biol 215:403-410, respectively. BLAST and BLAST 2.0
are used, with the parameters described herein, to determine
percent sequence identity or percent sequence similarity for the
nucleic acids of the invention. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information (http: //www.ncbi.nlm.nih.gov/). This
algorithm involves first identifying high scoring sequence pairs
(HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always>0) and N (penalty score for
mismatching residues; always<0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, M=5, N=-4 and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a wordlength of
3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see,
Henikoff & Henikoff, (1989) Proc Natl Acad Sci USA 89:10915)
uses alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a
comparison of both strands. Again, as with other suitable
algorithms, the stringency of comparison can be increased until the
program identifies only sequences that are more closely related to
those in the sequence listings herein (i.e., SEQ ID NOS:1-18,
rather than sequences that are more closely related to other
similar sequences such as, e.g., those nucleic acid sequences
represented by GENSEQ reference numbers: N91042, T77193, Q43524,
Q53550, N60156, and Q43525; by GenBank accession nos.:
K03104.1,.times.03922.1, NC.sub.--001347.1, and X17403.1; or by
other similar molecules found in any public database. (The GenBank
accession nos. for the first four GENSEQ sequences are: A01321,
AR094363, AR050546, and AR050544.) In other words, the stringency
of comparison of the algorithms can be increased so that all known
sequences are excluded.
[0060] The BLAST algorithm also performs a statistical analysis of
the similarity or identity between two sequences (see, e.g., Karlin
& Altschul, (1993) Proc Natl Acad Sci USA 90:5873-5787). One
measure of similarity or identity provided by the BLAST algorithm
is the smallest sum probability (P(N)), which provides an
indication of the probability by which a match between two
nucleotide or amino acid sequences would occur by chance. For
example, a nucleic acid is considered similar to a reference
sequence if the smallest sum probability in a comparison of the
test nucleic acid to the reference nucleic acid is less than about
0.2, more preferably less than about 0.01, and most preferably less
than about 0.001.
[0061] Another example of a useful algorithm is PILEUP. PILEUP
creates a multiple sequence alignment from a group of related
sequences using progressive, pairwise alignments to show
relationship and percent sequence identity or percent sequence
similarity. It also plots a tree or dendogram showing the
clustering relationships used to create the alignment. PILEUP uses
a simplification of the progressive alignment method of Feng &
Doolittle, (1987) J Mol Evol 35:351-360. The method used is similar
to the method described by Higgins & Sharp, (1989) CABIOS
5:151-153. The program can align up to 300 sequences, each of a
maximum length of 5,000 nucleotides or amino acids. The multiple
alignment procedure begins with the pairwise alignment of the two
most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences
are aligned by a simple extension of the pairwise alignment of two
individual sequences. The final alignment is achieved by a series
of progressive, pairwise alignments. The program is run by
designating specific sequences and their amino acid or nucleotide
coordinates for regions of sequence comparison and by designating
the program parameters. Using PILEUP, a reference sequence is
compared to other test sequences to determine the percent sequence
identity (or percent sequence similarity) relationship using the
following parameters: default gap weight (3.00), default gap length
weight (0.10), and weighted end gaps. PILEUP can be obtained from
the GCG sequence analysis software package, e.g., version 7.0
(Devereaux et al., (1984) Nuc Acids Res 12:387-395).
[0062] Another preferred example of an algorithm that is suitable
for multiple DNA and amino acid sequence alignments is the CLUSTALW
program (Thompson, J. D. et al., (1994) Nuc Acids Res
22:4673-4680). CLUSTALW performs multiple pairwise comparisons
between groups of sequences and assembles them into a multiple
alignment based on homology. Gap open and Gap extension penalties
were 10 and 0.05 respectively. For amino acid alignments, the
BLOSUM algorithm can be used as a protein weight matrix (Henikoff
and Henikoff, (1992) Proc Natl Acad Sci USA 89:10915-10919).
[0063] It will be understood by one of ordinary skill in the art,
that the above discussion of search and alignment algorithms also
applies to identification and evaluation of polynucleotide
sequences, with the substitution of query sequences comprising
nucleotide sequences, and where appropriate, selection of nucleic
acid databases.
[0064] Numbering of a given amino acid polymer or nucleotide
polymer "corresponds to numbering" of a selected amino acid polymer
or nucleic acid polymer when the position of any given polymer
component (e.g., amino acid residue, nucleotide residue) is
designated by reference to the same or an equivalent residue
position in the selected amino acid or nucleotide polymer, rather
than by the actual position of the component in the given polymer.
Thus, for example, the numbering of a given amino acid position in
a given polypeptide sequence corresponds to the same or equivalent
amino acid position in a selected polypeptide sequence used as a
reference sequence.
[0065] Another indication that two nucleic acid sequences are
substantially identical is that the two molecules hybridize to each
other under stringent conditions. The phrase "hybridizing
specifically to", refers to the binding, duplexing, or hybridizing
of a molecule only to a particular nucleotide sequence under
stringent conditions when that sequence is present in a complex
mixture (e.g., total cellular) DNA or RNA. "Bind(s) substantially"
refers to complementary hybridization between a probe nucleic acid
and a target nucleic acid and embraces minor mismatches that can be
accommodated by reducing the stringency of the hybridization media
to achieve the desired detection of the target polynucleotide
sequence. An extensive guide to the hybridization of nucleic acids
is found in Tijssen (1993) Laboratory Techniques in Biochemistry
and Molecular Biology--Hybridization with Nucleic Acid Probes, part
I, chapter 2, "Overview of principles of hybridization and the
strategy of nucleic acid probe assays," (Elsevier, New York), as
well as in Ausubel, supra. Hames and Higgins (1995) Gene Probes 1,
IRL Press at Oxford University Press, Oxford, England (Hames and
Higgins 1) and Hames and Higgins (1995) Gene Probes 2, IRL Press at
Oxford University Press, Oxford, England (Hames and Higgins 2)
provide details on the synthesis, labeling, detection and
quantification of DNA and RNA, including oligonucleotides.
[0066] "Stringent hybridization and wash conditions" in the context
of nucleic acid hybridization experiments, such as Southern and
northern hybridizations, are sequence dependent, and are different
under different environmental parameters. An extensive guide to the
hybridization of nucleic acids is found in Tijssen (1993), supra,
and in Hames and Higgins 1 and Hames and Higgins 2, supra.
[0067] For purposes of the present invention, generally, "highly
stringent" hybridization and wash conditions are selected to be
about 5.degree. C. or less lower than the thermal melting point
(T.sub.m) for the specific sequence at a defined ionic strength and
pH (as noted below, highly stringent conditions can also be
referred to in comparative terms). The T.sub.m is the temperature
(under defined ionic strength and pH) at which 50% of the test
sequence hybridizes to a perfectly matched probe. Very stringent
conditions are selected to be equal to the T.sub.m for a particular
probe.
[0068] The T.sub.m is the temperature of the nucleic acid duplexes
indicates the temperature at which the duplex is 50% denatured
under the given conditions and its represents a direct measure of
the stability of the nucleic acid hybrid. Thus, the T.sub.m
corresponds to the temperature corresponding to the midpoint in
transition from helix to random coil; it depends on length,
nucleotide composition, and ionic strength for long stretches of
nucleotides.
[0069] After hybridization, unhybridized nucleic acid material can
be removed by a series of washes, the stringency of which can be
adjusted depending upon the desired results. Low stringency washing
conditions (e.g., using higher salt and lower temperature) increase
sensitivity, but can product nonspecific hybridization signals and
high background signals. Higher stringency conditions (e.g., using
lower salt and higher temperature that is closer to the
hybridization temperature) lowers the background signal, typically
with only the specific signal remaining. See, Rapley, R. and
Walker, J. M. eds., Molecular Biomethods Handbook (Humana Press,
Inc. 1998) (hereinafter "Rapley and Walker"), which is incorporated
herein by reference in its entirety for all purposes.
[0070] The T.sub.m of a DNA-DNA duplex can be estimated using the
following equation:
T.sub.m(.degree. C.)=81.5.degree. C.+16.6(log.sub.10M)+0.41(%
G+C)-0.72(% f)-500/n
[0071] where M is the molarity of the monovalent cations (usually
Na+), (% G+C) is the percentage of guanosine (G) and cystosine (C )
nucleotides, (% f) is the percentage of formamide and n is the
number of nucleotide bases (i.e., length) of the hybrid. See,
Rapley and Walker, supra.
[0072] The T.sub.m of an RNA-DNA duplex can be estimated as
follows:
T.sub.m(.degree. C.)=79.8.degree. C.+18.5(log.sub.10M)+0.58(%
G+C)-11.8(% G+C).sup.20.56(% f)-820/n
[0073] where M is the molarity of the monovalent cations (usually
Na+), (% G+C)is the percentage of guanosine (G) and cystosine (C)
nucleotides, (% f) is the percentage of formamide and n is the
number of nucleotide bases (i.e., length) of the hybrid. Id.
[0074] Equations 1 and 2 are typically accurate only for hybrid
duplexes longer than about 100-200 nucleotides. Id.
[0075] The Tm of nucleic acid sequences shorter than 50 nucleotides
can be calculated as follows:
T.sub.m(.degree. C.)=4(G+C)+2(A+T)
[0076] where A (adenine), C, T (thymine), and G are the numbers of
the corresponding nucleotides.
[0077] An example of stringent hybridization conditions for
hybridization of complementary nucleic acids which have more than
100 complementary residues on a filter in a Southern or northern
blot is 50% formamide (or formalin) with 1 mg of heparin at
42.degree. C., with the hybridization being carried out overnight.
An example of stringent wash conditions is a 0.2.times. SSC wash at
65.degree. C. for 15 minutes (see Sambrook, supra for a description
of SSC buffer). Often the high stringency wash is preceded by a low
stringency wash to remove background probe signal. An example low
stringency wash is 2.times. SSC at 40.degree. C. for 15
minutes.
[0078] In general, a signal to noise ratio of 2.5.times.-5.times.
(or higher) than that observed for an unrelated probe in the
particular hybridization assay indicates detection of a specific
hybridization. Detection of at least stringent hybridization
between two sequences in the context of the present invention
indicates relatively strong structural similarity or homology to,
e.g., the nucleic acids of the present invention provided in the
sequence listings herein.
[0079] As noted, "highly stringent" conditions are selected to be
about 5.degree. C. or less lower than the thermal melting point
(T.sub.m) for the specific sequence at a defined ionic strength and
pH. Target sequences that are closely related or identical to the
nucleotide sequence of interest (e.g., "probe") can be identified
under highly stringency conditions. Lower stringency conditions are
appropriate for sequences that are less complementary. See, e.g.,
Rapley and Walker, supra.
[0080] Comparative hybridization can be used to identify nucleic
acids of the invention, and this comparative hybridization method
is a preferred method of distinguishing nucleic acids of the
invention. Detection of highly stringent hybridization between two
nucleotide sequences in the context of the present invention
indicates relatively strong structural similarity/homology to,
e.g., the nucleic acids disclosed herein. Highly stringent
hybridization between two nucleotide sequences demonstrates a
degree of similarity or homology of structure, nucleotide base
composition, arrangement or order that is greater than that
detected by stringent hybridization conditions. In particular,
detection of highly stringent hybridization in the context of the
present invention indicates strong structural similarity or
structural homology (e.g., nucleotide structure, base composition,
arrangement or order) to, e.g., the nucleic acids provided in the
sequence listings herein. For example, it is desirable to identify
test nucleic acids which hybridize to the exemplar nucleic acids
herein under stringent conditions.
[0081] Thus, one measure of stringent hybridization is the ability
to hybridize to one of the listed nucleic acids (e.g., nucleic acid
sequences SEQ ID NO: 1 to SEQ ID NO: 18, and complementary
polynucleotide sequences and fragments thereof) under highly
stringent conditions (or very stringent conditions, or ultra-high
stringency hybridization conditions, or ultra-ultra high stringency
hybridization conditions). Stringent hybridization (including,
e.g., highly stringent, ultra-high stringency, or ultra-ultra high
stringency hybridization conditions) and wash conditions can easily
be determined empirically for any test nucleic acid.
[0082] For example, in determining highly stringent hybridization
and wash conditions, the hybridization and wash conditions are
gradually increased (e.g., by increasing temperature, decreasing
salt concentration, increasing detergent concentration and/or
increasing the concentration of organic solvents, such as formalin,
in the hybridization or wash), until a selected set of criteria are
met. For example, the hybridization and wash conditions are
gradually increased until a probe comprising one or more nucleic
acid sequences selected from SEQ ID NO:1 to SEQ ID NO:18, and
complementary polynucleotide sequences and fragments thereof, binds
to a perfectly matched complementary target (again, a nucleic acid
comprising one or more nucleic acid sequences selected from SEQ ID
NO:1 to SEQ ID NO:18, and complementary polynucleotide sequences
and fragments thereof), with a signal to noise ratio that is at
least 2.5.times., and optionally 5.times. or more as high as that
observed for hybridization of the probe to an unmatched target. In
this case, the unmatched target is a nucleic acid corresponding to,
e.g., a known CMV promoter/enhancer homologue, e.g., a CMV
promoter/enhancer homologue homologue nucleic acid (other than
those in the accompanying sequence listing) that is present in a
public database such as GenBank.TM. at the time of filing of the
subject application. Examples of such unmatched target nucleic
acids include, e.g., nucleic acid sequences represented by GENSEQ
reference numbers: N91042, T77193, Q43524, Q53550, N60156, Q43525;
by GenBank accession nos.: K03104.1,.times.03922.1,
NC.sub.--001347.1, X17403.1; or by other similar molecules found in
any public database. (The GenBank accession nos. for the first four
GENSEQ sequences are: A01321, AR094363, AR050546, AR050544.)
[0083] A test nucleic acid is said to specifically hybridize to a
probe nucleic acid when it hybridizes at least 1/2 as well to the
probe as to the perfectly matched complementary target, i.e., with
a signal to noise ratio at least 1/2 as high as hybridization of
the probe to the target under conditions in which the perfectly
matched probe binds to the perfectly matched complementary target
with a signal to noise ratio that is at least about
2.5.times.-10.times., typically 5.times.-10.times. as high as that
observed for hybridization to any of the unmatched target nucleic
acids.
[0084] Ultra high-stringency hybridization and wash conditions are
those in which the stringency of hybridization and wash conditions
are increased until the signal to noise ratio for binding of the
probe to the perfectly matched complementary target nucleic acid is
at least 10 .times. as high as that observed for hybridization to
any of the unmatched target nucleic acids. A target nucleic acid
which hybridizes to a probe under such conditions, with a signal to
noise ratio of at least 1/2 that of the perfectly matched
complementary target nucleic acid is said to bind to the probe
under ultra-high stringency conditions.
[0085] Similarly, even higher levels of stringency can be
determined by gradually increasing the hybridization and/or wash
conditions of the relevant hybridization assay. For example, those
in which the stringency of hybridization and wash conditions are
increased until the signal to noise ratio for binding of the probe
to the perfectly matched complementary target nucleic acid is at
least 10.times., 20.times., 50.times., 100.times., or 500.times. or
more as high as that observed for hybridization to any of the
unmatched target nucleic acids. A target nucleic acid which
hybridizes to a probe under such conditions, with a signal to noise
ratio of at least 1/2 that of the perfectly matched complementary
target nucleic acid is said to bind to the probe under
ultra-ultra-high stringency conditions.
[0086] Target nucleic acids which hybridize to the nucleic acids
represented by SEQ ID NO: 1 to SEQ ID NO: N and complementary
polynucleotide sequences and fragments thereof under high,
ultra-high and ultra-ultra high stringency conditions are a feature
of the invention.
[0087] For distinguishing between duplexes with sequences of less
than about 100 nucleotides, a TMAC1 hybridization procedure known
to those of ordinary skill in the art can be used. See, e.g., Sorg,
U. et al. 1 Nucleic Acids Res. (Sep. 11, 1991) 19(17), incorporated
herein by reference in its entirety for all purposes.
[0088] "Substantially the entire length of a polynucleotide
sequence" or "substantially the entire length of a polypeptide
sequence" refers to at least about 50%, generally at least about
60%, 70%, or 75%, usually at least about 80%, or typically at least
about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, 99.5% or more of a length of a polynucleotide
sequence or polypeptide sequence.
[0089] A "polypeptide sequence" is a polymer of amino acids (a
protein, polypeptide, etc., comprising amino acid residues) or a
character string representing an amino acid polymer, depending on
context. Given the degeneracy of the genetic code, one or more
nucleic acids, or the complementary nucleic acids thereof, that
encode a specific polypeptide sequence can be determined from the
polypeptide sequence.
[0090] A "fragment" or "subsequence" is any portion of an entire
polynucleotide or polypeptide sequence. Thus, a "subsequence"
refers to a sequence of nucleic acids or amino acids that comprises
a part of a longer sequence of nucleic acids (e.g., polynucleotide)
or amino acids (e.g., polypeptide) respectively. In one aspect, the
invention provides a nucleic acid comprising a fragment that
comprises a unique subsequence in a nucleic acid selected from SEQ
ID NO:1 to SEQ ID NO:18 or complementary polynucleotide sequence or
a fragment thereof. The unique subsequence is unique as compared to
subsequences of any of the nucleic acid sequences represented by
GENSEQ reference numbers: N91042, T77193, Q43524, Q53550, N60156,
Q43525; by GenBank accession nos.: K03104.1,.times.03922.1,
NC.sub.--001347.1,.times.17403.1; or by other similar molecules
found in any public database or complementary polynucleotide
sequences thereof. (The GenBank accession nos. for the first four
GENSEQ sequences are: A01321, AR094363, AR050546, AR050544.) Such
unique subsequences can be determined by aligning any of SEQ ID NO:
1 to SEQ ID NO: N or corresponding complementary sequences or
fragments against the complete set of nucleic acids available,
e.g., in a public database, at the filing date of the subject
application. Alignment can be performed using the BLAST algorithm
set to default parameters. Any unique subsequence is useful, e.g.,
as a probe to identify the nucleic acids of the invention.
[0091] A nucleic acid, protein, peptide, polypeptide, or other
component is "isolated" when it is partially or completely
separated from components with which it is normally associated
(other peptides, polypeptides, proteins (including complexes, e.g.,
polymerases and ribosomes which may accompany a native sequence),
nucleic acids, cells, synthetic reagents, cellular contaminants,
cellular components, etc.), e.g., such as from other components
with which it is normally associated in the cell from which it was
originally derived. A nucleic acid, polypeptide, or other component
is isolated when it is partially or completely recovered or
separated from other components of its natural environment such
that it is the predominant species present in a composition,
mixture, or collection of components (i.e., on a molar basis it is
more abundant than any other individual species in the
composition). In preferred embodiments, the preparation consists of
more than about 70% or 75%, typically more than about 80%, or
preferably more than about 90% of the isolated species.
[0092] In one aspect, a "substantially pure" or "isolated" nucleic
acid (e.g., RNA or DNA), polypeptide, protein, or composition also
means where the object species (e.g., nucleic acid or polypeptide)
comprises at least about 50, 60, or 70 percent by weight (on a
molar basis) of all macromolecular species present. A substantially
pure or isolated composition can also comprise at least about 80,
90, or 95 percent by weight of all macromolecular species present
in the composition. An isolated object species can also be purified
to essential homogeneity (contaminant species cannot be detected in
the composition by conventional detection methods) wherein the
composition consists essentially of derivatives of a single
macromolecular species. The term "purified" generally denotes that
a nucleic acid, polypeptide, or protein gives rise to essentially
one band in an electrophoretic gel. It typically means that the
nucleic acid, polypeptide, or protein is at least about 50% pure,
60% pure, 70% pure, 75% pure, more preferably at least about 85%
pure, and most preferably at least about 99% pure.
[0093] The term "isolated nucleic acid" may refer to a nucleic acid
(e.g., DNA or RNA) that is not immediately contiguous with both of
the sequences with which it is immediately contiguous (i.e., one at
the 5' and one at the 3' end) in the naturally occurring genome of
the organism from which the nucleic acid of the invention is
derived. Thus, this term includes, e.g., a cDNA or a genomic DNA
fragment produced by polymerase chain reaction (PCR) or restriction
endonuclease treatment, whether such cDNA or genomic DNA fragment
is incorporated into a vector, integrated into the genome of the
same or a different species than the organism, including, e.g., a
virus, from which it was originally derived, linked to an
additional coding sequence to form a hybrid gene encoding a
chimeric polypeptide, or independent of any other DNA sequences.
The DNA may be double-stranded or single-stranded, sense or
antisense.
[0094] The term "recombinant" when used with reference, e.g., to a
cell, vector, nucleic acid, or polypeptide typically indicates that
the cell, vector, nucleic acid or polypeptide has been modified by
the introduction of a heterologous (or foreign) nucleic acid or the
alteration of a native nucleic acid, or that the polypeptide has
been modified by the introduction of a heterologous amino acid, or
that the cell is derived from a cell so modified. Recombinant cells
express nucleic acid sequences (e.g., genes) that are not found in
the native (non-recombinant) form of the cell or express native
nucleic acid sequences (e.g., genes) that would be abnormally
expressed, under-expressed, or not expressed at all. The term
"recombinant" when used with reference to a cell indicates that the
cell replicates a heterologous nucleic acid, or expresses a peptide
or protein encoded by a heterologous nucleic acid. Recombinant
cells can contain genes that are not found within the native
(non-recombinant) form of the cell. Recombinant cells can also
contain genes found in the native form of the cell wherein the
genes are modified and re-introduced into the cell by artificial
means. The term also encompasses cells that contain a nucleic acid
endogenous to the cell that has been modified without removing the
nucleic acid from the cell; such modifications include those
obtained by gene replacement, site-specific mutation, and related
techniques.
[0095] The terms "recombinant polynucleotide" or a "recombinant
polypeptide" encompass a non-naturally occurring polynucleotide or
polypeptide that includes nucleic acid or amino acid sequences,
respectively, from more than one source nucleic acid or
polypeptide, which source nucleic acid or polypeptide can be a
naturally occurring nucleic acid or polypeptide, or can itself have
been subjected to mutagenesis or other type of modification. A
nucleic acid or polypeptide may be deemed "recombinant" when it is
artificial or engineered, or derived from an artificial or
engineered polypeptide or nucleic acid. A recombinant nucleic acid
(e.g., DNA or RNA) can be made by the combination (e.g., artificial
combination) of at least two segments of sequence that are not
typically included together, not typically associated with one
another, or are otherwise typically separated from one another. A
recombinant nucleic acid can comprise a nucleic acid molecule
formed by the joining together or combination of nucleic acid
segments from different sources and/or artificially synthesized. A
"recombinant polypeptide" (or "recombinant protein") often refers
to a polypeptide (or protein) that results from a cloned or
recombinant nucleic acid or gene. The source polynucleotides or
polypeptides from which the different nucleic acid or amino acid
sequences are derived are sometimes homologous (i.e., have, or
encode a polypeptide that encodes, the same or a similar structure
and/or function), and are often from different isolates, serotypes,
strains, species, of organism or from different disease states, for
example.
[0096] The term "recombinantly produced" refers to an artificial
combination usually accomplished by either chemical synthesis
means, recursive sequence recombination of nucleic acid segments or
other diversity generation methods (such as, e.g., shuffling) of
nucleotides, or manipulation of isolated segments of nucleic acids,
e.g., by genetic engineering techniques known to those of ordinary
skill in the art. "Recombinantly expressed" typically refers to
techniques for the production of a recombinant nucleic acid in
vitro and transfer of the recombinant nucleic acid into cells in
vivo, in vitro, or ex vivo where it may be expressed or
propagated.
[0097] "Naturally occurring" as applied to an object refers to the
fact that the object can be found in nature as distinct from being
artificially produced by man. For example, a polypeptide or
polynucleotide sequence that is present in an organism (including
viruses, bacteria, protozoa, insects, plants or mammalian tissue)
that can be isolated from a source in nature and that has not been
intentionally modified by man in the laboratory is naturally
occurring. A "non-naturally occurring" object is one that is not
found in nature or is found in nature in a different form.
[0098] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
instance, a promoter or enhancer is operably linked to a coding
sequence if it directs or increases the transcription of the coding
sequence. A nucleic acid is said to "promote the expression" of an
operably linked coding sequence if the nucleic acid acts as a
promoter (i.e., direct transcription) or as an enhancer (i.e.,
increases transcription). "Operably linked" means that the DNA
sequences being linked are typically contiguous and, where
necessary to join two protein coding regions, contiguous and in
reading frame. However, since enhancers generally function when
separated from the promoter by several kilobases and intronic
sequences may be of variable lengths, some polynucleotide elements
may be operably linked but not contiguous.
[0099] A "recombinant expression cassette" or simply an "expression
cassette" is a nucleic acid construct, generated recombinantly or
synthetically, with operably linked nucleic acid elements that are
capable of effecting expression of a structural gene in hosts
compatible with such sequences. Expression cassettes include at
least a promoter and optionally, a transcription termination
signal. Typically, the recombinant expression cassette includes a
nucleic acid to be transcribed (e.g., a nucleic acid encoding a
desired polypeptide), which is termed a "transgene," and a
promoter. Additional factors necessary or helpful in effecting
expression may also be used as described herein. For example, an
expression cassette can also include nucleotide sequences that
encode a signal sequence that directs secretion of an expressed
protein from the host cell. Enhancers, and other nucleic acid
sequences that influence gene expression, can also be included in
an expression cassette.
[0100] An "exogenous" nucleic acid," "exogenous DNA segment,"
"heterologous sequence," or "heterologous nucleic acid," as used
herein, is one that originates from a source foreign to the
particular host cell, or, if from the same source, is modified from
its original form. Thus, a heterologous gene in a host cell
includes a gene that is endogenous to the particular host cell, but
has been modified. The terms refer to a DNA segment which is
foreign or heterologous to the cell, or homologous to the cell but
in a position within the host cell nucleic acid in which the
element is not ordinarily found. Exogenous DNA segments are
expressed to yield exogenous polypeptides.
[0101] A vector is a component or composition for facilitating cell
transduction, transfection, or infection by a selected nucleic
acid, or expression of the nucleic acid in the cell. Vectors
include, e.g., plasmids, cosmids, viruses, YACs, bacteria,
poly-lysine, etc. An "expression vector" is a nucleic acid
construct or sequence, generated recombinantly or synthetically,
with a series of specific nucleic acid elements that permit
transcription of a particular nucleic acid in a host cell. The
expression vector can be part of a plasmid, virus, or nucleic acid
fragment. The expression vector typically includes a nucleic acid
to be transcribed (i.e., a transgene) operably linked to a
promoter. The nucleic acid to be transcribed is typically under the
direction or control of the promoter.
[0102] Variants of the exemplary nucleic acids described herein can
be selected or screened for nucleic acids with or which confer
desirable properties, such as the ability to promote expression of
an operably linked transgene at a desired level. The term
"screening" describes, in general, a process that identifies
optimal molecules of the present invention, such as, e.g., the
novel promoters, fragments and homologues thereof, and related
expression cassettes and vectors. For screening and selection,
these molecules are linked to or include a transgene that encodes a
conveniently measured marker polypeptide. Other marker polypeptides
that can be used in selection and screening include, for example,
those that bind to a receptor, and/or induce or inhibit a desired
biological response in a test system or an in vitro, ex vivo or in
vivo application (e.g., induce or inhibit a T-cell proliferation
response). Selection is a form of screening in which identification
and physical separation are achieved simultaneously by expression
of a selection marker, which, in some genetic circumstances, allows
cells expressing the marker to survive while other cells die (or
vice versa). Screening markers include, for example, luciferase,
beta-galactosidase and green fluorescent protein, and the like.
Selection markers include drug and toxin resistance genes, and the
like. Although spontaneous selection can and does occur in the
course of natural evolution, in the present methods, selection is
performed by man.
[0103] A "specific binding affinity" between two molecules, e.g., a
ligand and a receptor, means a preferential binding of one molecule
for another in a mixture of molecules. The binding of the molecules
can be considered specific if the binding affinity is about
1.times.10.sup.4 M.sup.-1 to about 1.times.10.sup.7 M.sup.-1 (i.e.,
about 10.sup.-4-10.sup.-7 M) or greater.
[0104] The term "subject" as used herein includes, but is not
limited to, an organism, such as a mammal, including, e.g., a
human, non-human primate (e.g., baboon, orangutan, monkey), mouse,
pig, cow, goat, cat, rabbit, rat, guinea pig, hamster, horse,
monkey, sheep, or other non-human mammal; a non-mammal, including,
e.g., a non-mammalian vertebrate, such as a bird (e.g., a chicken
or duck) or a fish, and a non-mammalian invertebrate.
[0105] The term "pharmaceutical composition" means a composition
suitable for pharmaceutical use in a subject, including an animal
or human. A pharmaceutical composition generally comprises an
effective amount of an active agent and a carrier, including, e.g.,
a pharmaceutically acceptable carrier.
[0106] The term "effective amount" means a dosage or amount
sufficient to produce a desired result. The desired result may
comprise an objective or subjective improvement in the subject
receiving the dosage or amount.
[0107] A "prophylactic treatment" is a treatment administered to a
subject who does not display signs or symptoms of a disease,
pathology, or medical disorder, or displays only early signs or
symptoms of a disease, pathology, or disorder, such that treatment
is administered for the purpose of diminishing, preventing, or
decreasing the risk of developing the disease, pathology, or
medical disorder. A prophylactic treatment functions as a
preventative treatment against a disease or disorder. A
"prophylactic activity" is an activity of an agent, such as a
nucleic acid, vector, gene, polypeptide, protein, substance, or
composition thereof that, when administered to a subject who does
not display signs or symptoms of a pathology, disease or disorder,
or who displays only early signs or symptoms of a pathology,
disease, or disorder, diminishes, prevents, or decreases the risk
of the subject developing the pathology, disease, or disorder. This
effect is termed a "prophylactic effect."
[0108] A "prophylactically useful" agent or compound (e.g., nucleic
acid or polypeptide) refers to an agent or compound that is useful
in diminishing, preventing, treating, or decreasing development of
a pathology, disease or disorder.
[0109] A "therapeutic treatment" is a treatment administered to a
subject who displays symptoms or signs of a pathology, disease, or
disorder, in which treatment is administered to the subject for the
purpose of diminishing or eliminating those signs or symptoms of
the pathology, disease, or disorder. A "therapeutic activity" is an
activity of an agent, such as a nucleic acid, vector, gene,
polypeptide, protein, substance, or composition thereof, that
eliminates or diminishes signs or symptoms of a pathology, disease
or disorder, when administered to a subject suffering from such
signs or symptoms. This effect is termed a "therapeutic effect." A
"therapeutically useful" agent or compound (e.g., nucleic acid or
polypeptide) indicates that an agent or compound is useful in
diminishing, treating, or eliminating such signs or symptoms of a
pathology, disease or disorder.
[0110] An "immunogen" refers to a substance capable of provoking an
immune response, and includes, e.g., antigens, autoantigens that
play a role in induction of autoimmune diseases, and
tumor-associated antigens expressed on cancer cells. An immune
response of any type to an immunogen is termed an "immunogenic
effect." An "immunomodulatory molecule" refers to a substance
capable of altering an immune response provoked by an
immunogen.
[0111] An "antigen" refers to a substance that is capable of
eliciting the formation of antibodies in a host or generating a
specific population of lymphocytes reactive with that substance.
Antigens are typically macromolecules (e.g., proteins and
polysaccharides) that are foreign to the host.
[0112] An "adjuvant" refers to a substance that enhances an
antigen's immune-stimulating properties or the pharmacological
effect of a drug. For example, "Freund's Complete Adjuvant" is an
emulsion of oil and water containing an immunogen, an emulsifying
agent and mycobacteria. Another example, "Freund's incomplete
adjuvant," is the same but without mycobacteria.
[0113] The term "cytokine" includes, for example, interleukins,
interferons, chemokines, hematopoietic growth factors, tumor
necrosis factors and transforming growth factors. In general these
are small molecular weight proteins that regulate maturation,
activation, proliferation, and differentiation of the cells of the
immune system.
[0114] Generally speaking, a "co-stimulatory molecule" refers to a
molecule that acts in association or conjunction with, or is
involved with, a second molecule or with respect to an immune
response in a co-stimulatory pathway. In one aspect, a
co-stimulatory molecule may be an immunomodulatory molecule that
acts in association or conjunction with, or is involved with,
another molecule to stimulateor enhanceIn another aspect, a
co-stimulatory molecule is an immunomodulatory molecule that acts
in association or conjunction with, or is involved with, another
molecule toinhibit or suppress an immune response. A an immune
response. co-stimulatory molecule need not act simultaneously with,
or by the same mechanism, as the second molecule. Exemplary
co-stimulatory molecules include, e.g., B7-1 (CD80) and B7-2 (CD86)
polypeptide ligands, which are expressed on antigen-presenting
cells and act with an antigen in the stimulation of a T cell
receptor to effectuate an immune response. Additional
co-stimulatory molecules include CD54 or CD50 (ICAM), CD11a/18
(LFA-1) CD40, and ICOS (B7-H) which are also expressed on
antigen-presenting cells. Other co-stimulatory polypeptides
include, respectively, polypeptides that bind CD28 and/or CTLA-4
receptors on T cells (see, e.g., copending, commonly assigned US
Patent Application Ser. No. ______, entitled "Novel Co-Stimulatory
Molecules," filed Jun. 21, 2001 as LJAQ Attorney Docket No.
02-106720US (169.310US).
[0115] Generally, the nomenclature used hereafter and the
laboratory procedures in cell culture, molecular genetics,
molecular biology, nucleic acid chemistry, and protein chemistry
described below are those well known and commonly employed by those
of ordinary skill in the art. Standard techniques, such as
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., 1989 (hereinafter "Sambrook") and Current
Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current
Protocols, a joint venture between Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc. (1994, supplemented through
1999) (hereinafter "Ausubel"), are used for recombinant nucleic
acid methods, nucleic acid synthesis, cell culture methods, and
transgene incorporation, e.g., electroporation, injection, and
lipofection. Generally, oligonucleotide synthesis and purification
steps are performed according to specifications. The techniques and
procedures are generally performed according to conventional
methods in the art and various general references which are
provided throughout this document. The procedures therein are
believed to be well known to those of ordinary skill in the art and
are provided for the convenience of the reader.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0116] A. In General
[0117] The major immediate-early (IE) region transcriptional
regulatory elements, including promoter and enhancer sequences (the
promoter/enhancer region), of cytomegalovirus is widely used for
regulating transcription of genes, because it is highly active in a
broad range of host cell types (see, e.g., Foecking et al., Gene
45:101-105 (1986)). The human CMV promoter/enhancer region has been
found to be a strong promoter/enhancer (Boshart et al., Cell
41:521-530 (1985); Thomsen et al., Proc Natl Acad Sci 81:659-663
(1984)), including in transient expression systems (Foecking et
al., Gene 45:101-105 (1986); Pasleau et al., Gene 38:227-232
(1985)). The human CMV promoter has been shown to be a source of
transcriptional signal elements for expression of a variety of
heterologous proteins (Cockett et al., Biotechnology 8:662-667
(1990); Eaton et al., Biochemistry 25:8343-8353 (1986)).
[0118] Because the CMV promoter and enhancer are active in human
and animal cells, the improved (optimized) CMV promoter/enhancer
elements can be used to express foreign genes, including antigens,
such as, e.g., the cancer antigen EpCam/KSA and recombinant forms
thereof. Other examples of cancer antigens that can be expressed
using the promoter/enhancer elements of the invention include,
e.g., bullous pemphigoid antigen 2, prostate mucin antigen (PMA)
(Beckett and Wright (1995) Int. J. Cancer 62:703-710), tumor
associated Thomsen-Friedenreich antigen (Dahlenborg et al. (1997)
Int. J. Cancer 70:63-71), prostate-specific antigen (PSA) (Dannull
and Belldegrun (1997) Br. J. Urol. 1:97-103), luminal epithelial
antigen (LEA.135) of breast carcinoma and bladder transitional cell
carcinoma (TCC) (Jones et al. (1997) Anticancer Res. 17:685-687),
cancer-associated serum antigen (CASA) and cancer antigen 125 (CA
125) (Kierkegaard et al. (1995) Gynecol. Oncol. 59:251-254), the
epithelial glycoprotein 40 (EGP40) (Kievit et al. (1997) Intl. J.
Cancer 71:237-245), squamous cell carcinoma antigen (SCC) (Lozza et
al. (1997) Anticancer Res. 17: 525-529), cathepsin E (Mota et al.
(1997) Am. J. Pathol. 150:1223-1229), tyrosinase in melanoma
(Fishman et al. (1997) Cancer 79: 1461-1464), cell nuclear antigen
(PCNA) of cerebral cavemomas (Notelet et al. (1997) Surg. Neurol.
47: 364-370), DF3/MUC1 breast cancer antigen (Apostolopoulos et al.
(1996) Immunol. Cell. Biol. 74: 457-464; Pandey et al. (1995)
Cancer Res. 55: 4000-4003), carcinoembryonic antigen (Paone et al.
(1996) J. Cancer Res. Clin. Oncol. 122:499-503; Schlom et al.
(1996) Breast Cancer Res. Treat. 38:27-39), tumor-associated
antigen CA 19-9 (Tolliver and O'Brien (1997) South Med. J.
90:89-90; Tsuruta et al. (1997) Urol. Intl. 58:20-24), human
melanoma antigens MART-1/Melan-A27-35 and gplOO (Kawakami and
Rosenberg (1997) Intl. Rev. Immunol. 14:173-192; Zajac et al.
(1997) Intl. J. Cancer 71:491-496), the T and Tn pancarcinoma (CA)
glycopeptide epitopes (Springer (1995) Crit. Rev. Oncog. 6:57-85),
a 35 kD tumor-associated autoantigen in papillary thyroid carcinoma
(Lucas et al. (1996) Anticancer Res. 16:2493-2496), KH-1
adenocarcinoma antigen (Deshpande and Danishefsky (1997) Nature
387:164-166), the A60 mycobacterial antigen (Maes et al. (1996) J.
Cancer Res. Clin. Oncol. 122:296-300), heat shock proteins (HSPs)
(Blachere and Srivastava (1995) Semin. Cancer Biol. 6:349-355), and
MAGE, tyrosinase, melan-A and gp75 and mutant oncogene products
(e.g., p53, ras, CDk4, and HER-2/neu (Bueler and Mulligan (1996)
Mol. Med. 2:545-555; Lewis and Houghton (1995) Semin. Cancer Biol.
6: 321-327; Theobald et al. (1995) Proc. Nat'l. Acad. Sci. USA 92:
11993-11997), prostate specific membrane antigen (PSMA) Bangma CH
et al. (2000) Microsc Res Tech 51:430-5, TAG-72, McGuinness RP et
al. Hum Gene Ther (1999) 10:165-73, and variants, derivatives, and
mutated, and recombinant forms (e.g., shuffled forms) thereof of
these antigens.
[0119] The promoter/enhancer elements can also be used to express
co-stimulatory molecules, including, e.g., B7-1 and B7-2 ligands,
CD54 or CD50 (ICAM), CD11a/18 (LFA-1) CD40, and ICOS (B7-H). Other
co-stimulatory polypeptides include, respectively, polypeptides
that bind CD28 and/or CTLA-4 receptors on T cells (see, e.g.,
copending, commonly assigned U.S. patent application Ser. No.
______, entitled "Novel Co-Stimulatory Molecules," filed Jun. 21,
2001 as LJAQ Attorney Docket No. 02-106720US (169.310 US). The
promoter/enhancer elements can also be use to express adjuvants,
etc. In all of these embodiments, the improved (optimized) CMV
promoter/enhancer elements can be used both in animal and human
models and in a variety of applications, including therapeutic and
prophylactic treatment methods described herein.
[0120] The ability to control gene expression, especially mammalian
gene expression, is of particular importance to the success of
genetic vaccination and gene therapy, protein-based vaccines and
immunotherapy treatments, and also in the production in culture of
therapeutic and prophylactic polypeptides and proteins useful for
treatment methods or other applications.
[0121] In preferred embodiments, the present invention provides for
improved, optimized CMV transcriptional regulatory elements,
generated by recursive sequence recombination methods, such as,
e.g., DNA shuffling, which provide for optimized levels of gene
expression (including, e.g., expression of genes encoding antigens,
co-stimulatory molecules, adjuvants, etc.), and/or direct long-term
and regulatable transgene expression. The desired (optimized) level
of gene expression can be a significantly increased expression
(high-level expression), an slightly increased expression
(intermediate-level expression), or a reduced or low expression
(low- or reduced-level expression), wherein each such level is
compared, e.g., to a known or wild-type CMV molecule comprising
such regulatory elements). The desired level of gene expression
depends upon the particular need or application. Promoter sequences
that are optimal for any given application can be identified by
screening libraries of chimeric nucleic acids produced as described
herein using criteria suitable for the intended application.
[0122] For example, optimized promoters that produce increased
levels of expression and direct long-term and regulatable transgene
expression would be particularly useful in genetic (DNA)
vaccination, other immunostimulatory applications, and therapeutic
and prophylactic methods, since they would likely improve the
efficacy of such applications. In genetic vaccination methods, a
genetic vaccine vector expresses a gene sequence encoding an
antigen or adjuvant, which elicits or potentiates an immune
response.
[0123] Generally, in standard genetic vaccination applications
described previously, an insufficient amount of antigen is
expressed for effective treatment. An optimized promoter having an
ability to express a greater amount of one or more antigens and/or
adjuvants may be preferred depending on the particular therapeutic
or prophylactic treatment objective (e.g., for treatment of a viral
infection, such as hepatitis B or C infection, or of other
infectious diseases; chronic diseases, especially those in which an
enhanced immune response is desired; or a particular cancer).
[0124] In other genetic vaccination applications where, e.g., the
particular antigen of interest causes too strong an immune response
or is too active in the subject in which it is expressed (with
possibly lethal or adverse effects), a promoter of the invention
optimized to express a lower or intermediate level of antigen
(compared, e.g., to a known promoter, such as a hCMV promoter) can
be prepared and used be used with the antigen or adjuvant (e.g., in
an expression vector format comprising the optimized promoter
operably linked to a nucleic acid sequence encoding the antigen or
adjuvant of interest) so as to avoid the deleterious or unwanted
consequences.
[0125] In some applications, the concentration of each of one or
more antigens, adjuvants, or prophylactic or therapeutic agents is
important. For example, in immunotherapy methods employing
co-stimulatory molecules, the relative concentrations of these
molecules is important, since the concentration of one such
molecule may affect the concentration of another. For example, it
is often desirable to express low or intermediate level
concentrations of one or more co-stimulatory molecules (compared,
e.g., to expression levels induced by known or standard promoters,
such as hCMV promoters). The promoter can thus be optimized to
direct the expression of one or more co-stimulatory molecules in a
particular application.
[0126] In some applications, as, e.g., in certain DNA vaccines, it
may desirable to employ an expression vector comprising a weaker
promoter (e.g., a promoter optimized to direct a low- or
intermediate-level of expression of a sequence encoding an
antigen). For example, it may be desired to induce tolerance to a
specific protein expressed by the gene by employing a series of
separately administered, increasing doses of an antigen expressed
by a DNA vaccine. Thus, it may be beneficial to initiate genetic
allergy vaccination with low doses, as is done in conventional
immunotherapy. Notably, simply reducing the amount of plasmid DNA
injected may not be sufficient, because only few cells expressing
the allergy antigen may be sufficient to induce anaphylactic
reactions. Therefore, promoters of the invention having a range of
activities are likely to be useful in the dose escalation of
genetic allergy vaccines. A series of allergy antigen expression
vectors, each comprising one or more promoters that induce
different levels of antigen expression in vivo, can be employed
with successive inoculations (over time) in an allergy treatment
program to regulate antigen dose. The amount of allergen expressed
is thus boosted with each application for improved efficacy.
[0127] In some therapeutic or prophylactic applications, such as,
e.g., in a preventive or therapeutic DNA vaccine for a particular
cancer, it may be desirable to have a continued or prolonged amount
of an antigen, immunomodulatory, or co-stimulatory molecule
expressed in the subject being treated. For example, a nucleic acid
of the invention that expresses a co-stimulatory molecule, such as
a B7-1 or B7-2 molecule, or a variant thereof, or a polypeptide
that binds or selectively binds to either or both of the CD28
receptor or CTLA-4 receptor, can be targeted to tumor cells. The
promoter used in such DNA vaccine can be optimized for the
particular application using the methods and compositions of the
invention.
[0128] B. Chimeric Promoter/Enhancers
[0129] The present invention provides nucleic acids including novel
chimeric promoter/enhancers that are useful for expressing genes in
a variety of eukaryotic cells, including mammalian cells, and in in
vivo or ex vivo applications (including transplantation methods).
The promoter/enhancers find use in producing proteins for
commercial or other use, gene therapy, genetic vaccinations, and
many other uses.
[0130] 1. Nucleic Acids
[0131] The nucleic acids of the invention are generally capable of
promoting the expression of an operably linked transgene.
Accordingly, the nucleic acids of the invention typically comprises
a transcription start site and related sequences (e.g., a "TATA
box" and/or a "CAAT" or "CAAAT" box), which can be derived from a
CMV promoter sequence or a variant thereof or from a non-CMV
promoter sequence. In the latter case, a nucleic acid sequence of
the invention includes one or more other CMV sequences (e.g.,
enhancer sequences) or variants thereof operably linked to the
transcription start site.
[0132] Preferred nucleic acids of the invention include the
chimeric promoter/enhancer sequences disclosed herein (SEQ ID NOS:
1-18) as well as complementary polynucleotide sequences thereof.
However, the invention also comprises fragments of these
polynucleotide sequences, as well as variants including an
insertion, substitution, and/or deletion of one or more nucleotides
and nucleic acids that are otherwise modified. Preferably,
fragments, nucleotides sequence variants, and modified forms of the
disclosed polynucleotide sequences (collectively termed "CMV
promoter/enhancer variants" for ease of discussion) retain the
ability to promote the expression of an operably linked
transgene.
[0133] In one embodiment, variants of SEQ ID NOS:1-18 can be
designed based on the properties disclosed herein for these
polynucleotides. Thus, for example, the 12C9 polynucleotide
sequence (SEQ ID NO:3) lacks CMV promoter nucleic acid residues
beyond about nucleotide residue 909, numbered according to the
consensus sequence shown in FIG. 8. Yet this polynucleotide
sequence still serves as an efficient promoter of
.beta.-galactosidase expression as demonstrated by the in vivo
assay for anti-.beta.-galactosidase antibody shown in FIG. 6A. This
observation indicates that CMV promoter/enhancer sequences
downstream (relative to the direction of transcription) of the
residue corresponding to residue 909 in the FIG. 8 consensus
sequence are not required for efficient expression of an operably
linked transgene. Accordingly, the invention encompasses nucleic
acids that include variants of SEQ ID NOS: 1, 2, and 4-18 that lack
such downstream CMV promoter/enhancer sequences. In preferred
embodiments, such variants include the CAAT box and/or the TATA box
(both of these motifs are underlined in FIG. 8E) present in region
corresponding to about nucleotide residues 840-890 of the consensus
sequence shown in FIG. 8. Exemplary nucleic acids of this type lack
CMV promoter nucleic acid residues beyond about nucleotide residue
900, 910, 920, 930, and 940, numbered according to this consensus
sequence.
[0134] The polynucleotide sequences shown in FIG. 8 include a first
exon beginning at about nucleotide residue 810 and extending to
about nucleotide residue 932, numbered according to the consensus
sequence shown in FIG. 8. In some application, it may be desirable
to delete this sequence. Thus, invention also encompasses nucleic
acids that include variants of SEQ ID NOS: 1, 2, and 4-18 lacking
these exon sequences. Exemplary nucleic acids of this type lack CMV
promoter nucleic acid residues beyond about nucleotide residue 810,
820, 830, 840,850, 860, 870, 880, and 890, numbered according to
this consensus sequence.
[0135] Other variants of the disclosed sequences will be apparent
to the skilled practitioner in light of the guidance provided
herein. The design and production of such CMV promoter/enhancer
variants can be carried out using any of a wide variety of
diversity generating and/or mutational methods that are available
and described in the art, followed by screening or selection of
variants for desired properties. The procedures can be used
separately, and/or in combination to produce one or more variants
of a nucleic acid or set of nucleic acids. Individually and
collectively, these procedures provide robust, widely applicable
ways of generating diversified nucleic acids and sets of nucleic
acids (including, e.g., nucleic acid libraries) useful, e.g., for
the engineering or rapid evolution of CMV promoter/enhancer
variants derived from the polynucleotide sequences disclosed
herein.
[0136] Descriptions of a variety of diversity generating procedures
for generating nucleic acid variants are found in the following
publications and the references cited therein: Soong, N. et al.
(2000) "Molecular breeding of viruses" Nat Genet 25(4):436-439;
Stemmer, et al. (1999) "Molecular breeding of viruses for targeting
and other clinical properties" Tumor Targeting 4:1-4; Ness et al.
(1999) "DNA Shuffling of subgenomic sequences of subtilisin" Nature
Biotechnology 17:893-896; Chang et al. (1999) "Evolution of a
cytokine using DNA family shuffling" Nature Biotechnology
17:793-797; Minshull and Stemmer (1999) "Protein evolution by
molecular breeding" Current Opinion in Chemical Biology 3:284-290;
Christians et al. (1999) "Directed evolution of thymidine kinase
for AZT phosphorylation using DNA family shuffling" Nature
Biotechnology 17:259-264; Crameri et al. (1998)
[0137] "DNA shuffling of a family of genes from diverse species
accelerates directed evolution" Nature 391:288-291; Crameri et al.
(1997) "Molecular evolution of an arsenate detoxification pathway
by DNA shuffling," Nature Biotechnology 15:436-438; Zhang et al.
(1997) "Directed evolution of an effective fucosidase from a
galactosidase by DNA shuffling and screening" Proc. Natl. Acad.
Sci. USA 94:4504-4509; Patten et al. (1997) "Applications of DNA
Shuffling to Pharmaceuticals and Vaccines" Current Opinion in
Biotechnology 8:724-733; Crameri et al. (1996) "Construction and
evolution of antibody-phage libraries by DNA shuffling" Nature
Medicine 2:100-103; Crameri et al. (1996) "Improved green
fluorescent protein by molecular evolution using DNA shuffling"
Nature Biotechnology 14:315-319; Gates et al. (1996) "Affinity
selective isolation of ligands from peptide libraries through
display on a lac repressor `headpiece dimer`" Journal of Molecular
Biology 255:373-386; Stemmer (1996) "Sexual PCR and Assembly PCR"
In: The Encyclopedia of Molecular Biology. VCH Publishers, New
York. pp.447-457; Crameri and Stemmer (1995) "Combinatorial
multiple cassette mutagenesis creates all the permutations of
mutant and wildtype cassettes" BioTechniques 18:194-195; Stemmer et
al., (1995) "Single-step assembly of a gene and entire plasmid form
large numbers of oligodeoxy-ribonucleotides" Gene, 164:49-53;
Stemmer (1995) "The Evolution of Molecular Computation" Science
270: 1510; Stemmer (1995) "Searching Sequence Space" Bio/Technology
13:549-553; Stemmer (1994) "Rapid evolution of a protein in vitro
by DNA shuffling" Nature 370:389-391; and Stemmer (1994) "DNA
shuffling by random fragmentation and reassembly: In vitro
recombination for molecular evolution." Proc. Natl. Acad. Sci. USA
91:10747-10751.
[0138] Mutational methods of generating diversity include, for
example, site-directed mutagenesis (Ling et al. (1997) "Approaches
to DNA mutagenesis: an overview" Anal Biochem. 254(2): 157-178;
Dale et al. (1996) "Oligonucleotide-directed random mutagenesis
using the phosphorothioate method" Methods Mol. Biol. 57:369-374;
Smith (1985) "In vitro mutagenesis" Ann. Rev. Genet. 19:423-462;
Botstein & Shortle (1985) "Strategies and applications of in
vitro mutagenesis" Science 229:1193-1201; Carter (1986)
"Site-directed mutagenesis" Biochem. J. 237:1-7; and Kunkel (1987)
"The efficiency of oligonucleotide directed mutagenesis" in Nucleic
Acids & Molecular Biology (Eckstein, F. and Lilley, D. M. J.
eds., Springer Verlag, Berlin)); mutagenesis using uracil
containing templates (Kunkel (1985) "Rapid and efficient
site-specific mutagenesis without phenotypic selection" Proc. Natl.
Acad. Sci. USA 82:488-492; Kunkel et al. (1987) "Rapid and
efficient site-specific mutagenesis without phenotypic selection"
Methods in Enzymol. 154, 367-382; and Bass et al. (1988) "Mutant
Trp repressors with new DNA-binding specificities" Science
242:240-245); oligonucleotide-directed mutagenesis (Methods in
Enzymol. 100: 468-500 (1983); Methods in Enzymol. 154: 329-350
(1987); Zoller & Smith (1982) "Oligonucleotide-directed
mutagenesis using M13-derived vectors: an efficient and general
procedure for the production of point mutations in any DNA
fragment" Nucleic Acids Res. 10:6487-6500; Zoller & Smith
(1983) "Oligonucleotide-directed mutagenesis of DNA fragments
cloned into M13 vectors" Methods in Enzymol. 100:468-500; and
Zoller & Smith (1987) "Oligonucleotide-directed mutagenesis: a
simple method using two oligonucleotide primers and a
single-stranded DNA template" Methods in Enzymol. 154:329-350);
phosphorothioate-modified DNA mutagenesis (Taylor et al. (1985)
"The use of phosphorothioate-modified DNA in restriction enzyme
reactions to prepare nicked DNA" Nucl. Acids Res. 13: 8749-8764;
Taylor et al. (1985) "The rapid generation of
oligonucleotide-directed mutations at high frequency using
phosphorothioate-modified DNA" Nucl. Acids Res. 13: 8765-8787
(1985); Nakamaye & Eckstein (1986) "Inhibition of restriction
endonuclease Nci I cleavage by phosphorothioate groups and its
application to oligonucleotide-directed mutagenesis" Nucl. Acids
Res. 14: 9679-9698; Sayers et al. (1988) "Y-T Exonucleases in
phosphorothioate-based oligonucleotide-directed mutagenesis" Nucl.
Acids Res. 16:791-802; and Sayers et al. (1988) "Strand specific
cleavage of phosphorothioate-containing DNA by reaction with
restriction endonucleases in the presence of ethidium bromide"
Nucl. Acids Res. 16: 803-814); mutagenesis using gapped duplex DNA
(Kramer et al. (1984) "The gapped duplex DNA approach to
oligonucleotide-directed mutation construction" Nucl. Acids Res.
12: 9441-9456; Kramer & Fritz (1987) Methods in Enzymol.
"Oligonucleotide-directed construction of mutations via gapped
duplex DNA" 154:350-367; Kramer et al. (1988) "Improved enzymatic
in vitro reactions in the gapped duplex DNA approach to
oligonucleotide-directed construction of mutations" Nucl. Acids
Res. 16: 7207; and Fritz et al. (1988) "Oligonucleotide-directed
construction of mutations: a gapped duplex DNA procedure without
enzymatic reactions in vitro" Nucl. Acids Res. 16: 6987-6999).
[0139] CMV promoter/enhancer variants produced using one or more of
the methods herein, or otherwise available to one of skill, can be
selected or screened to determine whether the variation(s) confer
one or more desirable properties. This can include identifying any
activity that can be detected, for example, in an automated or
automatable format, by any of the assays in the art. In preferred
embodiments, CMV promoter/enhancer variants are screened in one or
more of the in vitro or in vivo assays described in the Examples.
Thus, variants can be operably linked to a conveniently measured
marker gene to form an expression cassette. Expression of the
marker gene can be detected, e.g., by FACS sorting to select for a
desired level of expression. Additional testing can be carried out
in vivo or in vitro to further characterize the variants and to
identify those have desired properties. A variety of related (or
even unrelated) properties can be evaluated, in serial or in
parallel, at the discretion of the practitioner.
[0140] The above-described diversity generating and/or mutational
methods can generate a plurality of different CMV promoter/enhancer
variants. Accordingly, the invention provides compositions
comprising at least two different nucleic acids of the invention.
Collections of different nucleic acids are typically termed
polynucleotide libraries, and such libraries are within the scope
of the invention, regardless of whether the nucleic acids are
present together in a composition or stored separately, e.g., in
separate bacterial colonies, separate vials of purified DNA,
etc.
[0141] The nucleic acids of the invention can provide a range of
different expression levels of an operably linked transgene. Thus,
in one embodiment, the nucleic acid includes a polynucleotide
sequence that promotes the expression of an operably linked
transgene at a level that is higher than the highest expression
level of the same transgene when operably linked to a nucleic acid
sequence corresponding to a human CMV promoter polynucleotide
sequence. In an alternative embodiment, the nucleic acid includes a
polynucleotide sequence that promotes the expression of an operably
linked transgene at a level that is lower than the lowest
expression level of the same transgene when operably linked to a
nucleic acid sequence corresponding to a human CMV promoter
polynucleotide sequence. The differences in expression level for
nucleic acids of the invention, as compared to human CMV promoter
sequences can be on the order of about 1.5-fold, 2-fold, 5-fold, or
10-fold or greater.
[0142] The nucleic acids of the invention, including those
specifically exemplified herein (e.g., SEQ ID NOS:1-18) and
fragments and variants thereof can all be produced and used as
described below. Thus, persons of skill in the art appreciate that
references herein to "chimeric CMV promoter/enhancers" or
"recombinant promoters" apply generally to all of the nucleic acids
of the invention (including fragment or variants) unless context
dictates otherwise.
[0143] 2. Production of Nucleic Acids
[0144] Nucleic acids of the invention can be prepared any of a
variety of methods well known to those of skill in the art. For
example, nucleic acids can be prepared by standard solid-phase
methods, according to standard synthetic methods. Typically,
fragments of up to about 100 bases are individually synthesized,
then joined (e.g., by enzymatic or chemical ligation methods, or
polymerase mediated recombination methods) to form essentially any
desired continuous sequence. For example, the nucleic acids of the
invention can be prepared by chemical synthesis using, e.g., the
classical phosphoramidite method described by Beaucage et al.,
(1981) Tetrahedron Letters 22:1859-69, or the method described by
Matthes et al., (1984) EMBO J. 3: 801-05., e.g., as is typically
practiced in automated synthetic methods. According to the
phosphoramidite method, oligonucleotides are synthesized, e.g., in
an automatic DNA synthesizer, purified, annealed, ligated and
cloned in appropriate vectors.
[0145] In addition, essentially any nucleic acid can be custom
ordered from any of a variety of commercial sources, such as The
Midland Certified Reagent Company (mcrc@oligos.com), The Great
American Gene Company (http://www.genco.com), ExpressGen Inc.
(www.expressgen.com), Operon Technologies Inc. (Alameda, Calif.)
and many others.
[0146] In some applications, it is advantageous to stabilize the
nucleic acid molecules described herein or to produce nucleic acid
molecules that are modified to better adapt them for particular
applications. To this end, the nucleic acid molecules of the
invention can contain phosphorothioates, phosphotriesters, methyl
phosphonates, short chain alkyl or cycloalkyl intersugar linkages
or short chain heteroatomic or heterocyclic intersugar ("backbone")
linkages. Most preferred are phosphorothioates and those with
CH2--NH--O--CH2, CH2--N(CH3)--O--CH2 (known as the
methylene(methylimino) or MMI backbone) and CH2--O--N(CH3)--CH2,
CH2--N(CH3)--N(CH3)--CH2, and O--N(CH3)--CH2--CH backbones (where
phosphodiester is O--P--O--CH2). Also preferred are nucleic acid
molecules having morpholino backbone structures. Summerton, J. E.
and Weller, D. D., U.S. Pat. No. 5,034,506. Other preferred
embodiments use a protein-nucleic acid or peptide-nucleic acid
(PNA) backbone, wherein the phosphodiester backbone of the nucleic
acid molecule is replaced with a polyamide backbone, the bases
being bound directly or indirectly to the aza nitrogen atoms of the
polyamide backbone. P. E. Nielsen, M. Egholm, R. H. Berg, O.
Buchardt, Science 1991, 254, 1497. Nucleic acid molecules of the
invention can contain alkyl and halogen-substituted sugar moieties
and/or can have sugar mimetics such as cyclobutyls in place of the
pentofuranosyl group. In other preferred embodiments, the nucleic
acid molecules can include at least one modified base form or
"universal base" such as inosine. Nucleic acid molecules can, if
desired, include an RNA cleaving group, a cholesteryl group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of the nucleic acid molecule, and/or a
group for improving the pharmacodynamic properties of the nucleic
acid molecule.
[0147] 3. Nucleic Acid Compositions
[0148] The invention also contemplates standard manipulations of
the nucleic acids of the invention and therefore includes
compositions that represent the intermediates or end-products of
standard recombinant DNA techniques. Thus, for example, the
invention includes a composition produced by the cleaving of one or
more the nucleic acids, e.g., by mechanical, chemical, or enzymatic
means. Examples of enzymes suitable for enzymatic cleavage include
a restriction endonuclease, an RNAse or a DNAse, and the like. The
invention also includes a composition produced by a process
comprising incubating one or more of the nucleic acids in the
presence of deoxyribonucleotide triphosphates and a nucleic acid
polymerase.
[0149] In an exemplary embodiment, the nucleic acid polymerase is a
thermostable polymerase, such as those useful in amplification
methods. Examples of in vitro amplification methods, including the
polymerase chain reaction (PCR) the ligase chain reaction (LCR),
Q.beta.-replicase amplification and other RNA polymerase mediated
techniques are found in Berger, Sambrook, and Ausubel, as well as
Mullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide
to Methods and Applications (Innis et al. eds.) Academic Press
Inc., San Diego, Calif. (1990) (Innis); Arnheim & Levinson
(Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991)
3:81-94; (Kwoh et al. (1989) Proc. Natl Acad. Sci. USA 86:1173;
Guatelli et al. (1990) Proc. Natl Acad. Sci. USA 87:1874; Lomell et
al. (1989) J. Clin. Chem. 35:1826; Landegren et al. (1988) Science
241:1077-1080; Van Brunt (1990) Biotechnology 8:291-294; Wu and
Wallace (1989) Gene 4:560; and Barringer et al. (1990) Gene 89:117.
Improved methods of cloning in vitro amplified nucleic acids are
described in Wallace et al., U.S. Pat. No. 5,426,039.
[0150] 4. Expression Cassettes
[0151] The invention provides expression cassettes in which a
chimeric promoter/enhancer polynucleotide sequence or fragment or
variant of the invention is typically situated adjacent to one or
more restriction sites at which one can insert a nucleic acid
(i.e., a transgene) to be expressed. The expression cassettes of
the invention optionally include transcription termination signals.
Additional factors necessary or helpful in effecting expression may
also be used as described herein. For example, an expression
cassette can also include nucleotide sequences that encode a signal
sequence that directs secretion of an expressed protein from the
host cell.
[0152] The chimeric promoter/enhancer polynucleotide sequences, or
fragments or variants thereof is joined to nucleic acids that are
to be expressed (e.g., coding regions for polypeptides, tRNA and
rRNA molecules, antisense nucleic acids, and the like), using
techniques that are known to those of skill in the art. Suitable
nucleic acids can encode a protein from any organism, e.g., a
viral, bacterial, eukaryotic, mammalian, or human protein. Viral
proteins of interest include those from dengue virus, human
immunodeficiency virus (HIV), Japanese encephalitis virus,
Venezuelan encephalitis virus. Examples of nucleic acids that can
be incorporated into an expression cassette of the invention
include a nucleic acid encoding: an immunogen; an immunomodulatory
molecule, such as a co-stimulatory molecule (e.g., B7-1, B7-2, or
other polypeptide that binds or associates with a CD28 and/or
CTLA-4 receptor); an antigen (e.g., a cancer antigen, such as
EpCam/KSA; hepatitus B surface antigen or fragment thereof;
antigens from hepatitis A, hepatitis C, etc.), including a
multivalent or cross-reactive antigen; an adjuvant; an allergen, an
antibody; a bacterial toxin, including, e.g., staph/strep
enterotoxin and CT/LT (choleratoxin, labile enterotoxin); a
cytokine or cytokine receptor (e.g., IL-10 antagonist or
receptor);and a prophylactic or therapeutic polypeptide. Other
exemplary nucleic acids that can be included in the expression
cassettes of the invention include those encoding any of a variety
proteins described in commonly assigned PCT Application No.
US99/03022 (WO 99/41369), entitled "Genetic Vaccine Vector
Engineering," filed February 10, 1999 (106.310WO); commonly
assigned PCT Application No. US99/03020 (WO 99/41368), entitled
"Optimization of Immunomodulatory Properties of Genetic Vaccines,"
filed on Feb. 10, 1999 (155.110WO); commonly assigned PCT
Application No. US99/03023 (WO 99/41402), entitled "Targeting of
Genetic Vaccine Vectors," filed on February 10, 1999
(156.110WO);
[0153] commonly assigned PCT Application No. US99/02944 (WO
99/41383), entitled "Antigen Library Immunization," filed on Feb.
10, 1999 (157.110WO); commonly assigned PCT Application No.
US97/17302 (WO 98/13485), entitled "Methods for Optimization of
Gene Therapy by Recursive Sequence Shuffling and Selection," filed
Sep. 26, 1997 (107.410WO); commonly assigned PCT Application No.
US00/16984 (WO 00/00234), entitled "Methods and Compositions for
Engineering of Attenuated Vaccines," filed Jun. 20, 2000
(133.110WO); each of which is incorporated herein by reference in
its entirety for all purposes.
[0154] A wide variety of cloning and in vitro amplification methods
suitable for the construction of recombinant nucleic acids such as
expression vectors are well-known to persons of ordinary skill in
the art. Examples of these techniques and instructions sufficient
to direct persons of skill through many cloning exercises are found
in Berger and Kimmel, Guide to Molecular Cloning Techniques,
Methods in Enzymology volume 152 Academic Press, Inc., San Diego,
Calif. (Berger); and Current Protocols in Molecular Biology, F. M.
Ausubel et al., eds., Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
(2000 Supplement) (Ausubel).
[0155] C. Vectors and Cells
[0156] The chimeric promoter/enhancers of the invention are useful
for the production of proteins from eukaryotic, particularly
mammalian, cell culture. As described above, the promoter/enhancers
are operably linked to a coding region for the polypeptide of
interest to form an expression cassette, which is introduced into
an expression vector. This construct is then introduced into the
cells to be used for production. Alternatively, the nucleic acids
of the invention can be introduced into a vector in the absence an
expression cassette. Such constructs are useful, for example, for
propagating nucleic acids of the invention as an alternative to the
synthetic methods described above.
[0157] In both types of constructs, the vector can, for example, be
a plasmid, a cosmid, a phage, a virus or fragment thereof, a
bacterial artificial chromosome (BAC), a yeast artificial
chromosome (YAC). Large numbers of suitable vectors and promoters
are known to those of skill in the art, and are commercially
available.
[0158] General texts which describe molecular biological techniques
useful herein, including the use of vectors, promoters and many
other relevant topics, include Berger and Kimmel, Guide to
Molecular Cloning Techniques, Methods in Enzymology volume 152
Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al.,
Molecular Cloning--A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989
("Sambrook") and Current Protocols in Molecular Biology, Ausubel et
al., eds., Current Protocols, a joint venture between Greene
Publishing Associates, Inc. and John Wiley & Sons, Inc.,
(supplemented through 2000) ("Ausubel")).
[0159] Once the chimeric promoter/enhancer of the invention is
inserted into a vector, the construct is introduced into the host
cells. Suitable host cells for expression of the recombinant
polypeptides are known to those of skill in the art, and include,
for example, eukaryotic cells including insect, mammalian and
fungal cells. In a preferred embodiment, Aspergillus niger is used
as the host cell. Transformation and infection methods for
mammalian and other cells are described in Berger and Ausubel,
supra. In some embodiments it is advantageous to introduce a
polynucleotide library of the invention into a population of host
cells, e.g., for propagation or expression and, optionally,
screening an/or selection of constructs for desired properties.
[0160] D. Recombinant Protein Production
[0161] In one embodiment, a population of cells comprising a
nucleic acid of the invention operably linked to a transgene
encoding a polypeptide is used for recombinant protein production.
Thus, the chimeric promoter/enhancers of the invention or fragments
or variants thereof can be used to express a transgene in
anyapplication in which expression of the encoded polypeptide is
desired. Examples include research applications, e.g., where the
polypeptide is expressed in functional studies; any application,
including in vitro or in vivo research or diagnositic assays, in
which expression of a marker polypeptide is desired. In vivo
applications, including gene therapy and genetic vaccination are
discussed in greater detail below. The nucleic acids of the
invention can also be used to produce any polypeptide of interest
for research, medical, or industrial use.
[0162] When it is desirable to isolate the polypeptide, the
polypeptide can be expressed in at least the subset of the
population of cells or progeny thereof, which are usually in
culture. Preferably the cells are cultured in a nutrient medium
under conditions in which the nucleic acid promotes expression of
the polypeptide. The culture conditions, such as temperature, pH
and the like, are those previously used with the host cell selected
for expression, and will be apparent to those skilled in the art
and in the references cited herein, including, e.g., Freshney
(1994) Culture of Animal Cells, a Manual of Basic Technique, third
edition, Wiley-Liss, New York and the references cited therein.
[0163] Any of a number of well-known techniques for large- or
small-scale production of proteins can be employed in expressing
the polypeptides of the invention. These include, but are not
limited to, the use of a shaken flask, a fluidized bed bioreactor,
a roller bottle culture system, and a stirred tank bioreactor
system. Cell culture can be carried out in a batch, fed-batch, or
continuous mode.
[0164] After sufficient polypeptide has been expressed, the
polypeptide is generally isolated or recovered from the cells or
from the nutrient medium. Methods for isolation or recovery of
recombinant proteins produced as described above are well-known and
vary depending on the expression system employed. A polypeptide
including a signal sequence can be recovered from the culture
medium or the periplasm. Polypeptides can also be expressed
intracellularly and recovered from cell lysates.
[0165] The expressed polypeptides can be purified from culture
medium or a cell lysate by any method capable of separating the
polypeptide from one or more components of the host cell or culture
medium. Typically, the polypeptide is separated from host cell
and/or culture medium components that would interfere with the
intended use of the polypeptide.
[0166] As a first step, the culture medium or cell lysate is
usually centrifuged or filtered to remove cellular debris. The
supernatant is then typically concentrated or diluted to a desired
volume or diafiltered into a suitable buffer to condition the
preparation for further purification.
[0167] The polypeptide can then be further purified using
well-known techniques.
[0168] The technique chosen will vary depending on the properties
of the expressed polypeptide.
[0169] If, for example, the polypeptide is expressed as a fusion
protein containing an affinity domain, purification typically
includes the use of an affinity column containing the cognate
binding partner. For instance, polypeptides fused with
hexahistidine or similar metal affinity tags can be purified by
fractionation on an immobilized metal affinity column.
[0170] One of skill in the art would recognize that after
biological expression, or purification, the polypeptides may
possess a conformation substantially different than the native
conformations of the constituent polypeptides. In this case, it may
be necessary to denature and reduce the polypeptide and then to
cause the polypeptide to re-fold into the preferred conformation.
Methods of reducing and denaturing proteins and inducing re-folding
are well known to those of skill in the art (See, Debinski et al.
(1993) J. Biol. Chem., 268:14065-14070; Kreitman and Pastan (1993)
Bioconjug. Chem., 4:581-585; and Buchner, et al., (1992) Anal.
Biochem., 205:263-270). Debinski et al., for example, describe the
denaturation and reduction of inclusion body proteins in
guanidine-DTE. The protein is then refolded in a redox buffer
containing oxidized glutathione and L-arginine.
[0171] In an alternative embodiment, cells comprising a nucleic
acid of the invention operably linked to a transgene encoding a
polypeptide are in vivo. For example, the nucleic acids of the
invention can be used to produce transgenic organisms that express
the encoded polypeptide in a tissues or byproduct, including a
bodily fluid, such as urine or milk. Any transgenic organism of
interest, in which the polypeptide is expressed for production,
research, or other purposes can be produced using conventional
techniques. Transgenic mammal are of particular interest and are
readily produced from mammalian cells selected, e.g., from
fertilized oocytes, embryonic stem cells, or pluripotent stem
cells. When the transgenic organism is used for protein production,
the expressed polypeptide is recovered from the transgenic organism
or byproduct and can optionally be isolation using standard protein
purification methods, including those described above.
[0172] E. Gene Therapy and Genetic Vaccination
[0173] In some embodiments, the promoter/enhancers of the invention
are used for gene therapy. For such applications, the
promoter/enhancers can be operably linked to a gene that is to be
expressed upon introduction into a cell. Broadly speaking, a gene
therapy vector is an exogenous polynucleotide which produces a
medically useful phenotypic effect upon the mammalian cell(s) into
which it is transferred. The chimeric promoter/enhancers of the
invention are also useful for use in genetic vaccination. For
example, the chimeric promoter/enhancers can be used to obtain
expression of an immunogenic polypeptide that is operably linked to
the promoter/enhancer. In such applications, a suitable nucleic
acid or vector of the invention can be introduced into cells in
culture, followed by introduction of the cells are subsequently
into the subject, i.e., ex vivo administration of the nucleic acid
or vector. Alternatively, the nucleic acid or vector can be
introduced into the cells of the subject by administering the
nucleic acid or vector directly to the subject. The choice of
vector (if used), formulation of the nucleic acid or vector, and
mode of administration will vary depending on the particular
application.
[0174] 1. Vectors
[0175] Vectors used in gene therapy and genetic vaccination can be
viral or nonviral. A vector may or may not have an origin of
replication. For example, it is useful to include an origin of
replication in a vector for propagation of the vector prior to
administration to a patient. However, the origin of replication can
often be removed before administration if the vector is designed to
integrate into host chromosomal DNA or bind to host mRNA or DNA.
Viral vectors are usually introduced into a patient as components
of a virus. Illustrative vectors include, for example,
adenovirus-based vectors (Cantwell (1996) Blood 88:4676-4683;
Ohashi (1997) Proc Natl Acad Sci USA 94:1287-1292), Epstein-Barr
virus-based vectors (Mazda (1997) J Immunol Methods 204:143-151),
adenovirus-associated virus vectors, Sindbis virus vectors (Strong
(1997) Gene Ther 4:624-627), herpes simplex virus vectors (Kennedy
(1997) Brain 120:1245-1259) and retroviral vectors (Schubert (1997)
Curr Eye Res 16:656-662).
[0176] Nonviral vectors, typically dsDNA, can be transferred as
naked DNA or associated with a transfer-enhancing vehicle, such as
a receptor-recognition protein, liposome, lipoamine, or cationic
lipid. This DNA can be transferred into a cell using a variety of
techniques well known in the art. For example, naked DNA can be
delivered by the use of liposomes which fuse with the cellular
membrane or are endocytosed, i.e., by employing ligands attached to
the liposome, or attached directly to the DNA, that bind to surface
membrane protein receptors of the cell resulting in endocytosis.
Alternatively, the cells may be permeabilized to enhance transport
of the DNA into the cell, without injuring the host cells. One can
use a DNA binding protein, e.g., HBGF-1, known to transport DNA
into a cell. These procedures for delivering naked DNA to cells are
useful in vivo. For example, by using liposomes, particularly where
the liposome surface carries ligands specific for target cells, or
are otherwise preferentially directed to a specific organ, one may
provide for the introduction of the DNA into the target
cells/organs in vivo.
[0177] The chimeric promoter/enhancers of the invention can also be
used for gene therapy in the absence of a vector. The DNA segments
that include the chimeric promoter/enhancer can be introduced into
cells using a system which targets the segments to the particular
gene that is to be expressed using the promoter/enhancer. Suitable
targeting technology is described in, for example, U.S. Pat. No.
6,054,288.
[0178] In some embodiments, the optimized recombinant promoters of
the invention are used in conjunction with a vector, including, for
example, an expression vector or genetic vaccine vector. The choice
of vector and each of its components, including, e.g., the one or
more recombinant promoters employed in the vector, one or more
antigens, and/or one or more co-stimulatory sequences, and the
like, can be optimized for the particular purpose of treating one
or more specific conditions, including, for example, allergy,
cancer, or other conditions. The choice of a chimeric
promoter/enhancer for a particular vector format can be based on a
particular functional activity, such as the degree of expression
desired of a vector component (e.g., a high-, low-, or
intermediate-activity promoter), the type of tissue in which the
promoter is to operate (tissue-specific promoter), or a
cell-specific regulated promoter that optimally drives
transcription in a desired cell type(s). In each instance, the
promoter can be optimized using recursive sequence recombination
and selection methods analogous to those described herein.
[0179] Vectors of the present invention comprising at least one
recombinant promoter of the present invention can be designed to
include one or more nucleic acid sequences that express one or more
modulators, immunomodulators, or immunostimulatory molecules.
Optimized immunomodulators, immunostimulatory molecules and methods
for obtaining optimized immunodulators and immunostimulatory
molecules are described in commonly assigned PCT Application No.
US99/03020 (WO 99/41368), entitled "Optimization of
Immunomodulatory Properties of Genetic Vaccines," and copending,
commonly assigned U.S. patent application Ser. No. ______, entitled
"Novel Co-Stimulatory Molecules," filed on Jun. 21, 2001 as LJAQ
Attorney Docket No. 02-106720US (169.31US), each of which is
incorporated herein by reference in its entirety for all purposes.
These optimized immunomodulatory or immunostimulatory sequences are
particularly suitable for use as components of the multicomponent
genetic vaccines of the invention. Multiple modulators can be
expressed from a monocistronic or multicistronic form of the
vector. One or more vectors comprising optimized promoters of the
invention can be used in conjunction with or as multicomponent
genetic vaccines, which are capable of tailoring an immune response
as is most appropriate to achieve a desired effect (see, e.g.,
commonly assigned PCT Application No. PCT/US99/03022 (WO 99/41369),
entitled "Genetic Vaccine Vector Engineering," which is
incorporated herein by reference in its entirety for all
purposes).
[0180] The vectors comprising recombinant promoters of the
invention can also be engineered to direct maximal synthesis and
release of one or more chemokines from the target cells, e.g., in a
desired ratio. Genetic vaccine components, and methods for
obtaining components, that provide optimal release of chemokines
are described in PCT Application No. US99/03020 (WO 99/41368).
[0181] The recombinant optimized promoters of the invention can
also be used in conjunction with optimized antigens. Types of
wild-type antigens that can be employed for various conditions and
for use in genetic vaccines are described in commonly assigned PCT
Application No. PCT/US99/02944 (WO 99/41383), entitled "Antigen
Library Immunization," which is incorporated herein by reference in
its entirety for all purposes. Furthermore, multiple antigens can
be expressed from a monocistronic or multicistronic form of the
vector comprising at least one recombinant promoter of the
invention. Moroever, an antigen for a particular condition can be
optimized using recombination and selection methods analogous to
those described herein. Such methods, and antigens appropriate for
various conditions, are described in PCT Application No.
PCT/US99/02944.
[0182] A vector engineered to direct a THI response is preferred
for many of the immune responses mediated by the antigens described
herein (see, e.g., PCT Application No. PCT/US99/03022). It is
sometimes advantageous to employ a genetic vaccine that is targeted
for a particular target cell type (e.g., an antigen presenting cell
or an antigen processing cell). Vector components for targeting
genetic vaccine vectors to particular cell types, and methods of
obtaining improved targeting, are described in commonly assigned
PCT Application No. US99/03023 (WO 99/41402), entitled "Targeting
of Vaccine Vectors," which is incorporated herein by reference in
its entirety for all purposes.
[0183] Genetic vaccines which include optimized vector modules,
including optimized promoters of the invention are useful for
treating many diseases and other conditions that are either
mediated by a mammalian immune system or are susceptible to
treatment by an appropriate immune response. Representative
examples of these diseases are listed in PCT Appn. No. US 99/03022
(WO 99/41369). Antigens appropriate for each are described in PCT
Application No. PCT/US99/02944 (WO 99/41383). Examples of genetic
vaccines within the scope of the invention include: prophylactic
vaccines for infectious diseases, including HIV, dengue, and HBV;
therapeutic vaccines for infectious diseases such as HBV, HIV, and
other major chronic infectious disease targets; therapeutic cancer
vaccines; therapeutic allergy vaccines; therapeutic vaccines for
autoimmune disease; vaccines that express, e.g., novel
immunomodulatory proteins that can be used to augment the immune
response as adjuvants or vaccine components. A preferred genetic
vaccine includes an expression vector including a recombinant
promoter of the invention that expresses both a co-stimulatory
molecule, such as, e.g., a CD28-binding protein, and an antigen,
such as a cancer antigen.
[0184] 2. Pharmaceutical Compositions and Methods of
Administration
[0185] Gene therapy and genetic vaccine vectors are useful for
treating and/or preventing various diseases and other conditions.
The following discussion focuses on the on the use of vectors
because gene therapy and genetic vaccine method typically employ
vectors, but persons of skill in the art appreciate that the
nucleic acids of the invention can, depending on the particular
application, be employed in the absence of vector sequences.
Accordingly, references in the following discussion to vectors
should be understood as also relating to nucleic acids of the
invention that lack vector sequences.
[0186] Vectors can be delivered to a subject to induce an immune
response or other therapeutic or prophylactic response. Suitable
subjects include, but are not limited to, a mammal, including,
e.g., a human, primate, monkey, orangutan, baboon, mouse, pig, cow,
cat, goat, rabbit, rat, guinea pig, hamster, horse, sheep; or a
non-mammalian vertebrate such as a bird (e.g., a chicken or duck)
or a fish, or invertebrate.
[0187] Vectors can be delivered in vivo by administration to an
individual patient, typically by local (direct) administration or
by systemic administration (e.g., intravenous, intraperitoneal,
intramuscular, subdermal, intracranial, anal, vaginal, oral, buccal
route or they can be inhaled) or they can be administered by
topical application. Alternatively, vectors can be delivered to
cells ex vivo, such as cells explanted from an individual patient
(e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or
universal donor hematopoietic stem cells, followed by
reimplantation of the cells into a patient, usually after selection
for cells which have incorporated the vector.
[0188] In local (direct) administration formats, the nucleic acid
or vector is typically administered or transferred directly to the
cells to be treated or to the tissue site of interest (e.g., tumor
cells, tumor tissue sample, organ cells, blood cells, cells of the
skin, lung, heart, muscle, brain, mucosae, liver, intestine,
spleen, stomach, lymphatic system, cervix, vagina, prostate, mouth,
tongue, etc.) by any of a variety of formats, including topical
administration, injection (e.g., by using a needle or syringe), or
vaccine or gene gun delivery, pushing into a tissue, organ, or skin
site. For standard gene gun administration, the vector or nucleic
acid of interest is precipitated onto the surface of microscopic
metal beads. The microprojectiles are accelerated with a shock wave
or expanding helium gas, and penetrate tissues to a depth of
several cell layers. For example, the AccelTM Gene Delivery Device
manufactured by Agacetus, Inc. Middleton Wis. is suitable for use
in this embodiment. The nucleic acid or vector can be delivered,
for example, intramuscularly, intradermally, subdermally,
subcutaneously, orally, intraperitoneally, intrathecally,
intravenously, or placed within a cavity of the body (including,
e.g., during surgery), or by inhalation or vaginal or rectal
administration.
[0189] In in vivo indirect contact/administration formats, the
nucleic acid or vector is typically administered or transferred
indirectly to the cells to be treated or to the tissue site of
interest, including those described above (such as, e.g., skin
cells, organ systems, lymphatic system, or blood cell system,
etc.), by contacting or administering the nucleic acid or vector of
the invention directly to one or more cells or population of cells
from which treatment can be facilitated. For example, tumor cells
within the body of the subject can be treated by contacting cells
of the blood or lymphatic system, skin, or an organ with a
sufficient amount of the polypeptide such that delivery of the
nucliec acid or vector to the site of interest (e.g., tissue,
organ, or cells of interest or blood or lymphatic system within the
body) occurs and effective prophylactic or therapeutic treatment
results. Such contact, administration, or transfer is typically
made by using one or more of the routes or modes of administration
described above.
[0190] A large number of delivery methods are well known to those
of skill in the art. Such methods include, for example
liposome-based gene delivery (Debs and Zhu (1993) WO 93/24640;
Mannino and Gould-Fogerite (1988) BioTechniques 6(7):682-691; Rose
U.S. Pat No. 5,279,833; Brigham (1991) WO 91/06309; and Felgner et
al. (1987) Proc. Natl Acad. Sci. USA 84:7413-7414), as well as use
of viral vectors (e.g., adenoviral (see, e.g., Berns et al. (1995)
Ann. NY Acad. Sci. 772:95-104; Ali et al. (1994) Gene Ther.
1:367-384; and Haddada et al. (1995) Curr. Top. Microbiol. Immunol.
199 (Pt 3):297-306 for review), papillomaviral, retroviral (see,
e.g., Buchscher et al. (1992) J. Virol. 66(5) 2731-2739; Johann et
al. (1992) J. Virol. 66 (5):1635-1640 (1992); Sommerfelt et al.,
(1990) Virol. 176:58-59; Wilson et al. (1989) J. Virol.
63:2374-2378; Miller et al., J. Virol. 65:2220-2224 (1991);
Wong-Staal et al., PCT/US94/05700, and Rosenburg and Fauci (1993)
in Fundamental Immunology, Third Edition Paul (ed) Raven Press,
Ltd., New York and the references therein, and Yu et al., Gene
Therapy (1994) supra.), and adeno-associated viral vectors (see,
West et al. (1987) Virology 160:38-47; Carter et al. (1989) U.S.
Pat. No. 4,797,368; Carter et al. WO 93/24641 (1993); Kotin (1994)
Human Gene Therapy 5:793-801; Muzyczka (1994) J. Clin. Invst.
94:1351 and Samulski (supra) for an overview of AAV vectors; see
also, Lebkowski, U.S. Pat. No. 5,173,414; Tratschin et al. (1985)
Mol. Cell. Biol. 5(11):3251-3260; Tratschin, et al. (1984) Mol.
Cell. Biol., 4:2072-2081; Hermonat and Muzyczka (1984) Proc. Natl
Acad. Sci. USA, 81:6466-6470; McLaughlin et al. (1988) and Samulski
et al. (1989) J. Virol., 63:03822-3828), and the like.
[0191] "Naked" DNA and/or RNA that comprises a genetic vaccine can
be introduced directly into a tissue, such as muscle, by injection
using a needle or other similar device. See, e.g., U.S. Pat. No.
5,580,859. Other methods such as "biolistic" or particle-mediated
transformation (see, e.g., Sanford et al., U.S. Pat. No. 4,945,050;
U.S. Pat. No. 5,036,006) are also suitable for introduction of
genetic vaccines into cells of a mammal according to the invention.
These methods are useful not only for in vivo introduction of DNA
into a subject, such as a mammal, but also for ex vivo modification
of cells for reintroduction into a mammal. DNA is conveniently
introduced directly into the cells of a mammal or other subject
using, e.g., injection, such as via a needle, or a "gene gun." As
for other methods of delivering genetic vaccines, if necessary,
vaccine administration is repeated in order to maintain the desired
level of immunomodulation, such as the level of T cell activation.
Alternatively, nucleotides can be impressed into the skin of the
subject.
[0192] Gene therapy and genetic vaccine vectors (e.g.,
adenoviruses, liposomes, papillomaviruses, retroviruses, etc.) can
be administered directly to the subject (usually a mammal) for
transduction of cells in vivo. The vectors can be formulated as
pharmaceutical compositions for administration in any suitable
manner, including parenteral (e.g., subcutaneous, intramuscular,
intradermal, or intravenous), topical, oral, rectal, vaginal,
intrathecal, buccal (e.g., sublingual), or local administration,
such as by aerosol or transdermally, for immunotherapeutic or other
prophylactic and/or therapeutic treatment. Pretreatment of skin,
for example, by use of hair-removing agents, may be useful in
transdermal delivery. Suitable methods of administering such
packaged nucleic acids are available and well known to those of
skill in the art, and, although more than one route can be used to
administer a particular composition, a particular route can often
provide a more immediate and more effective reaction than another
route.
[0193] Pharmaceutical compositions of the invention can, but need
not, include a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers are determined in part by the
particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there are a wide variety of suitable formulations of pharmaceutical
compositions of the present invention. A variety of aqueous
carriers can be used, e.g., buffered saline and the like. These
solutions are sterile and generally free of undesirable matter.
These compositions may be sterilized by conventional, well known
sterilization techniques. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions such as pH adjusting and
buffering agents, toxicity adjusting agents and the like, for
example, sodium acetate, sodium chloride, potassium chloride,
calcium chloride, sodium lactate and the like. The concentration of
gene therapy or genetic vaccine vector in these formulations can
vary widely, and will be selected primarily based on fluid volumes,
viscosities, body weight and the like in accordance with the
particular mode of administration selected and the patient's
needs.
[0194] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the packaged
nucleic acid suspended in diluents, such as water, saline or PEG
400; (b) capsules, sachets or tablets, each containing a
predetermined amount of the active ingredient, as liquids, solids,
granules or gelatin; (c) suspensions in an appropriate liquid; and
(d) suitable emulsions. Tablet forms can include one or more of
lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn
starch, potato starch, tragacanth, microcrystalline cellulose,
acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium,
talc, magnesium stearate, stearic acid, and other excipients,
colorants, fillers, binders, diluents, buffering agents, moistening
agents, preservatives, flavoring agents, dyes, disintegrating
agents, and pharmaceutically compatible carriers. Lozenge forms can
comprise the active ingredient in a flavor, usually sucrose and
acacia or tragacanth, as well as pastilles comprising the active
ingredient in an inert base, such as gelatin and glycerin or
sucrose and acacia emulsions, gels, and the like containing, in
addition to the active ingredient, carriers known in the art. It is
recognized that the gene therapy vectors and genetic vaccines, when
administered orally, must be protected from digestion. This is
typically accomplished either by complexing the vector with a
composition to render it resistant to acidic and enzymatic
hydrolysis or by packaging the vector in an appropriately resistant
carrier such as a liposome. Means of protecting vectors from
digestion are well known in the art. The pharmaceutical
compositions can be encapsulated, e.g., in liposomes, or in a
formulation that provides for slow release of the active
ingredient.
[0195] The packaged nucleic acids, alone or in combination with
other suitable components, can be made into aerosol formulations
(e.g., they can be "nebulized") to be administered via inhalation.
Aerosol formulations can be placed into pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen,
and the like.
[0196] Suitable formulations for rectal administration include, for
example, suppositories, which consist of the packaged nucleic acid
with a suppository base. Suitable suppository bases include natural
or synthetic triglycerides or paraffin hydrocarbons. In addition,
it is also possible to use gelatin rectal capsules which consist of
a combination of the packaged nucleic acid with a base, including,
for example, liquid triglycerides, polyethylene glycols, and
paraffin hydrocarbons.
[0197] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intradermal, subdermal, intraperitoneal, and
subcutaneous routes, include aqueous and non-aqueous, isotonic
sterile injection solutions, which can contain one or more
antioxidants, buffers, bacteriostats, and solutes that render the
formulation isotonic with the blood of the intended recipient, and
aqueous and non-aqueous sterile suspensions that can include
suspending agents, solubilizers, thickening agents, stabilizers,
and preservatives. In the practice of this invention, compositions
can be administered, for example, by intravenous infusion, orally,
topically, intraperitoneally, intravesically or intrathecally.
Parenteral administration and intravenous administration are the
preferred methods of administration. The formulations of packaged
nucleic acid can be presented in unit-dose or multi-dose sealed
containers, such as ampoules and vials.
[0198] Injection solutions and suspensions can be prepared from
sterile powders, granules, and tablets of the kind previously
described. Cells transduced by the packaged nucleic acid can also
be administered intravenously or parenterally.
[0199] The dose administered to a patient, in the context of the
present invention should be sufficient to effect a beneficial
effect, such as an immune or other prophylactic or therapeutic
response in the patient over time. The dose will be determined by
the efficacy of the particular vector employed and the condition of
the patient, as well as the body weight or vascular surface area of
the patient to be treated. The size of the dose also will be
determined by the existence, nature, and extent of any adverse
side-effects that accompany the administration of a particular
vector, or transduced cell type in a particular patient.
[0200] In determining the effective amount of the vector to be
administered in the treatment or prophylaxis of an infection or
other condition, the physician evaluates vector toxicities,
progression of the disease, and the production of anti-vector
antibodies, if any. In general, the dose equivalent of a naked
nucleic acid from a vector for a typical 70 kilogram patient can
range from about 10 ng to about 1 g, about 100 ng to about 100 mg,
about 1 .mu.g to about 10 mg, about 10 .mu.g to about 1 mg, or from
about 30-300 .mu.g. Doses of vectors used to deliver the nucleic
acid are calculated to yield an equivalent amount of therapeutic
nucleic acid. Administration can be accomplished via single or
divided doses.
[0201] In therapeutic applications, compositions are administered
to a patient suffering from a disease (e.g., an infectious disease
or autoimmune disorder) in an amount sufficient to cure or at least
partially arrest or ameliorate the disease or at least one of its
complications. An amount adequate to accomplish this is defined as
a "therapeutically effective dose." Amounts effective for this use
will depend upon the severity of the disease and the general state
of the patient's health. Single or multiple administrations of the
compositions may be administered depending on the dosage and
frequency as required and tolerated by the patient. In any event,
the composition should provide a sufficient quantity of protein to
effectively treat the patient.
[0202] In prophylactic applications, compositions are administered
to a human or other mammal to induce an immune or other
prophylactic response that can help protect against the
establishment of an infectious disease or other condition.
[0203] The toxicity and therapeutic efficacy of the vectors that
include chimeric promoter/enhancers provided by the invention are
determined using standard pharmaceutical procedures in cell
cultures or experimental animals. One can determine the LD.sub.50
(the dose lethal to 50% of the population) and the ED.sub.50 (the
dose therapeutically effective in 50% of the population) using
procedures presented herein and those otherwise known to those of
skill in the art.
[0204] A typical pharmaceutical composition for intravenous
administration would be about 0.1 to 10 mg per patient per day.
Dosages from 0.1 up to about 100 mg per patient per day may be
used, particularly when the drug is administered to a secluded site
and not into the blood stream, such as into a body cavity or into a
lumen of an organ. Substantially higher dosages are possible in
topical administration. For recombinant promoters of the invention
that express the linked transgene at high levels, it may be
possible to achieve the desired effect using lower doses, e.g., on
the order of about 1 .mu.g or 10 .mu.g per patient per day. Actual
methods for preparing parenterally administrable compositions will
be known or apparent to those skilled in the art and are described
in more detail in such publications as Remington's Pharmaceutical
Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania
(1980).
[0205] The vectors or nucleic acids that include the chimeric
promoter/enhancers of the invention can be packaged in packs,
dispenser devices, and kits for administering the vectors to a
mammal. For example, packs or dispenser devices that contain one or
more unit dosage forms are provided. Typically, instructions for
administration of the compounds will be provided with the
packaging, along with a suitable indication on the label that the
compound is suitable for treatment of an indicated condition. For
example, the label may state that the active compound within the
packaging is useful for treating a particular infectious disease,
autoimmune disorder, tumor, or for preventing or treating other
diseases or conditions that are mediated by, or potentially
susceptible to, a mammalian immune response.
[0206] F. Character Strings
[0207] The present invention provides computers, computer readable
media and integrated systems comprising character strings
corresponding to the sequence information herein for the nucleic
acids herein.
[0208] Various methods and genetic algorithms (GOs) known in the
art can be used to detect homology or similarity between different
character strings, or can be used to perform other desirable
functions such as to control output files, provide the basis for
making presentations of information including the sequences and the
like. Examples include BLAST, discussed supra. Extensive examples
of the use of sequences in silico are found in, e.g.,
PCTIUS00/01202 "METHODS FOR MAKING CHARACTER STRINGS,
POLYNUCLEOTIDES AND POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" by
Selifonov et al., filed Jan. 18, 2000; PCT/US00/01230
"OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION" by Crameri et
al., filed Jan. 18, 2000; and PCTIUS00/01138 "METHODS OF POPULATING
DATA STRUCTURES FOR USE IN EVOLUTIONARY SIMULATIONS" by Selifonov
and Stemmer, filed Jan. 18, 2000.
[0209] Thus, different types of homology and similarity of various
stringency and length can be detected and recognized in the
integrated systems herein. For example, many homology determination
methods have been designed for comparative analysis of sequences of
biopolymers, for spell-checking in word processing, and for data
retrieval from various databases. With an understanding of
double-helix pair-wise complement interactions among 4 principal
nucleobases in natural polynucleotides, models that simulate
annealing of complementary homologous polynucleotide strings can
also be used as a foundation of sequence alignment or other
operations typically performed on the character strings
corresponding to the sequences herein (e.g., word-processing
manipulations, construction of figures comprising sequence or
subsequence character strings, output tables, etc.). An example of
a software package with genetic algorithms for calculating sequence
similarity is BLAST, which can be adapted to the present invention
by inputting character strings corresponding to the sequences
herein.
[0210] Similarly, standard desktop applications such as word
processing software (e.g., Microsoft Word.TM. or Corel
WordPerfect.TM.) and database software (e.g., spreadsheet software
such as Microsoft Excel.TM., Corel Quattro PrO.TM., or database
programs such as Microsoft Access.TM. or Paradox.TM.) can be
adapted to the present invention by inputting a character string
corresponding to the nucleic acids of the invention. For example,
the integrated systems can include the foregoing software having
the appropriate character string information, e.g., used in
conjunction with a user interface (e.g., a GUI in a standard
operating system such as a Windows, Macintosh or LINUX system) to
manipulate strings of characters. As noted, specialized alignment
programs such as BLAST can also be incorporated into the systems of
the invention for alignment of nucleic acid (or corresponding
character strings).
[0211] Integrated systems for analysis in the present invention
typically include a digital computer with GO software for aligning
sequences, as well as data sets entered into the software system
comprising any of the sequences herein. The computer can be, e.g.,
a PC (Intel x86 or Pentium chip-compatible DOS.TM., OS2.TM.
WINDOWS.TM. WINDOWS NT.TM., WINDOWS95.TM., WINDOWS98.TM. LINUX
based machine, a MACINTOS.TM., Power PC, or a UNIX based (e.g.,
SUNTM work station) machine) or other commercially common computer
which is known to one of skill. Software for aligning or otherwise
manipulating sequences is available, or can easily be constructed
by one of skill using a standard programming language such as
Visualbasic, Fortran, Basic, Java, or the like.
[0212] Any controller or computer optionally includes a monitor
which is often a cathode ray tube ("CRT") display, a flat panel
display (e.g., active matrix liquid crystal display, liquid crystal
display), or others. Computer circuitry is often placed in a box
which includes numerous integrated circuit chips, such as a
microprocessor, memory, interface circuits, and others. The box
also optionally includes a hard disk drive, a floppy disk drive, a
high capacity removable drive such as a writeable CD-ROM, and other
common peripheral elements. Inputting devices such as a keyboard or
mouse optionally provide for input from a user and for user
selection of sequences to be compared or otherwise manipulated in
the relevant computer system.
[0213] The computer typically includes appropriate software for
receiving user instructions, either in the form of user input into
a set parameter fields, e.g., in a GUI, or in the form of
preprogrammed instructions, e.g., preprogrammed for a variety of
different specific operations. The software then converts these
instructions to appropriate language for instructing the operation
of the fluid direction and transport controller to carry out the
desired operation.
[0214] The software can also include output elements for
controlling nucleic acid synthesis (e.g., based upon a sequence or
an alignment of a sequence herein) or other operations which occur
downstream from an alignment or other operation performed using a
character string corresponding to a sequence herein.
[0215] In one embodiment, the invention provides an integrated
system comprising a computer or computer readable medium comprising
a database having one or more sequence records. Each of the
sequence records comprises one or more character strings
corresponding to a nucleic acid or polypeptide or protein sequence
selected from SEQ ID NO:1 to SEQ ID NO:18 or a fragment or variant
thereof. The integrated system further comprises a use input
interface allowing a use to selectively view the one or more
sequence records. In one such integrated system, the computer or
computer readable medium comprises an alignment instruction set
that aligns the character strings with one or more additional
character strings corresponding to a nucleic acid or polypeptide or
protein sequence.
[0216] One such integrated system includes an instruction set that
comprises at least one of the following: a local sequence
comparison or a local homology comparison determination, a sequence
alignment or a homology alignment determination, a sequence
identity or similarity search or a search for similarity
determination, a sequence identity or similarity determination, a
structural similarity search, a structure determination, a nucleic
acid motif determination, a hypothetical translation, a
determination of a restriction map, a sequence recombination and a
BLAST determination. In some embodiments, the system further
comprises a readable output element that displays an alignment
produced by the alignment instruction set.
[0217] Methods of using a computer system to present information
pertaining to at least one of a plurality of sequence records
stored in a database are also provided. Each of the sequence
records comprises at least one character string corresponding to
SEQ ID NO: 1 to SEQ ID NO:18 or a fragment or variant thereof. The
method comprises determining at least one character string
corresponding to one or more of these sequences or a subsequence
thereof; determining which of the at least one character string of
the list are selected by a user; and displaying each of the
selected character strings, or aligning each of the selected
character strings with an additional character string. The method
may further comprise displaying an alignment of each of the
selected character strings with an additional character string
and/or displaying the list.
EXAMPLES
[0218] The following examples are offered to illustrate, but not to
limit the present invention.
[0219] Materials and Methods
[0220] CMV isolates
[0221] Four strains of cytomegalovirus (CMV) were obtained from
American Type Culture Collection (ATCC) (Rockville, Md.). Human
AD169 (VR-538; Rowe W. (1956) Proc. Soc. Exp. Biol. Med.
145:794-801) and Human Towne (VR-977; Plotkin SA (1975) Infect.
Immun. 12:521-27) strains were isolated from human patients with
CMV infections, while the 68-1 (Asher D M (1969) Bacteriol. Proc.
269:91) and CSG (Black H (1963) Proc. Soc. Exp. Biol. Med. 112:601)
strains were isolated from Rhesus and Vervet monkeys,
respectively.
[0222] Propagation of CMV isolates in Culture
[0223] All CMV isolates were passaged by coculture with WI-38
cells, a human diploid fibroblast cell line also obtained from ATCC
(CCL-75; Hayflick L and Moorhead PS (1961) Exp. Cell Res.
25:585-621). Fibroblast monolayers were infected with CMV isolates
when they were .about.80% confluent. Following adsorption for 1
hour at 37.degree. C., DMEM with 5% FCS was added, and the cultures
incubated at 37.degree. C. Supernatants were collected when cell
monolayers showed extensive cytopathic effect, and cleared of cell
debris by centrifuging at 10 000.times. g for 10 min at 4.degree.
C. Clarified supernatants were stored at -80.degree. C. until
needed.
[0224] Purification of Viral DNA
[0225] Virus-containing supernatants were layered onto a sorbitol
cushion (20% D-sorbitol, 50 mM Tris [pH 7.2], 1 mM MgCl.sub.2) and
centrifuged at 55 000.times. g for 1 hour to pellet the virus.
Virions were resuspended in 2 mL of 50 mM Tris [pH 8.0]-1 mM
MgCl.sub.2, and an equal volume of lysis buffer (150 mM Tris [pH
8.0], 1 mM MgCl.sub.2, 0.2 mM EDTA, 200 mM NaCl, 1% sodium
sarkosyl, 200 .mu.g proteinase K per mL) was added. The lysate was
incubated at 37.degree. C. for 3 to 5 hours. Liberated viral DNA
was extracted four times by gently rocking with an equal volume of
phenol and chloroform (1:1; vol:vol). The DNA was extracted twice
more with chloroform and then precipitated with ethanol. The
precipitate was washed with 80% ethanol, air dried briefly, and
resuspended in TE (10 mM Tris [ph 8.0], 1 mM EDTA) overnight. Viral
DNAs were stored at -20.degree. C.
[0226] Amplification of CMV Promoter Sequences by PCR
[0227] CMV promoter sequences were amplified using the XL PCR kit
(Promega, Madison, Wis.) according to the manufacturer's protocol.
Primers used for amplifying the sequences included tails encoding
EcoR1 or BamH1 sites, allowing the PCR product to be digested with
these enzymes for cloning. The primers used were used to amplify
promoter sequences from human and monkey CMVs:
[0228] Primersforpromoters in Human CMV Strains Towne and
AD169:
1 5'-ATA GCA CTG AGA CCT ATC GAA TTC ATA TGA GGC TAT ATC GCC GAT
A-3' (SEQ ID NO:24) 5'-TCA GTG AAC GCT TAT CTA GGA TCC AAG GAC GGT
GAC TGC AGA AAA-3' (SEQ ID NO:25)
[0229] Primers for Rhesus Monkey CMV Promoter:
2 5'-ATA GCA CTG AGA CCT ATC GAA TTC AAT GGC GAC TTG GCA TTG AGC
CAA TT-3' (SEQ ID NO:26) 5'-ATA GCA CTG AGA CCT ATC GAA TTC ACT TGG
CAC GGT GCC AAG TTT-3' (SEQ ID NO:27) 5'-TCA GTG AAC GCT TAT CTA
GGA TCC TAT CCG CGT TCC AAT GCA CCC TT-3' (SEQ ID NO:28) 5'-TCA GTG
AAC GCT TAT CTA GGA TCC TAT CCG CAT TCC AAT GCA CCG T-3' (SEQ ID
NO:29)
[0230] For a description of the human CMV (hCMV) promoters, see,
e.g., U.S. Pat. No. 5,385,839 and Meier, J., et al., Intervirology
39:331-342 (1996), the full disclosure of which is incorporated
herein by reference in its entirety for all purposes. For cloning
procedure for a hCMV and Rhesus CMV promoter, see, e.g., U.S. Pat.
No. 5,385,839 and Alcendor et al., Virology 194:815-812 (1993), the
full disclosure of each of which is incorporated herein by
reference in its entirety for all purposes. The nucleotide
sequences for human CMV promoters, Towne and AD169 strains, are
shown in FIG. 8. The sequence for human CMV promoter Towne strain
is shown at GenBank Accession No. X03922. The nucleotide sequences
for the Rhesus and Vervet monkey CMV promoters are shown in FIG.
10. Rhesus CMV IE promoter is shown in Alcendor et al., Virology
194:815-812(1993). AGM CMV IE (Colburn strain) is shown at GenBank
Accession No. M16019.
[0231] Building a Vector for Screening Novel Chimeric Promoter
Sequences Resulting from Shuffling of CMV Promoter Sequences
("Chimeric Promoter Sequences")
[0232] The SR.alpha. promoter nucleic sequence (as described in
Tackebe, Y. et al., Molecular and Cellular Biol 8:466-472 (1988))
was amplified by PCR from plasmid AR11677 (for a description of
this plasmid, see Whitehom et al., Biotechnology 13:1215-1219
(1995), FIG. 1, termed "Alpha+KH/HPAP20") using the following two
primers encoding Age 1 restriction sites.
3 5'-ATA GCA CTG AGA CCTATC ACC GGT TGG TCC TGT AGT TTG CTA ACA
CA-3' (SEQ ID NO:30) 5'-TCA GTG AAC GCT TAT CTA ACC GGT TCG AGG CAG
CTT GGA TCT GTA ACG-3' (SEQ ID NO:31)
[0233] The resulting SR.alpha. promoter sequence fragment
(.about.950 bp) was digested with Age l, and cloned into the Agel
site of vector pEGFP-1 (Clontech; Palo Alto, Calif.) (enhanced
green fluorescent protein). A clone with this SR.alpha. promoter
sequence fragment in the forward orientation was revealed by
restriction enzyme digestion. This plasmid was named pEGFP-1
(SR.alpha.).
[0234] The monoclonal antibody 179 (mAb179) epitope nucleic acid
sequence was amplified by PCR from plasmid ARI1677 using the
following two primers encoding Age 1 and BsrG1 restriction enzyme
sites.
4 5'-ATT CTA CCA TGT CTC ACC GGT CGC CAC CAT GGC CTT ACC AGT GAG
CGC CTT GC-3' (SEQ ID NO:32) 5'-TCA CTA CCT AGT AGT TGT ACA GTA TCT
TAT CAT GTC TGG ATC A-3' (SEQ ID NO:33)
[0235] Following digestion with Age 1 and BsrG1 restriction
enzymes, the mAb179 epitope nucleic acid fragment was cloned into
Clontech pEGFP-1 using Age 1 and BsrG1 restriction sites, thereby
removing the EGFP (enhanced green fluorescent protein) gene from
the vector.
[0236] A fragment comprising the SR.alpha. promoter nucleic acid
sequence, EGFP gene sequence, and BGH poly A nucleic acid sequence
(the EGFP gene and BGH poly A sequences comprised part of the
pEGFP-1 Clontech vector, discussed above) was amplified by PCR from
plasmid pEGFP-1(SR.alpha.) using the following two primers encoding
Eco47111 and Xhol restriction enzyme sites.
5 5'-TGA GTG AAC GCT TAT CTA AGC GCT TTC TGT GGA ATG TGT GTC AGT
TA-3' (SEQ ID NO:34) 5'-ATA GCA CTG AGA CCT ATC CTC GAG TAC GCC TTA
AGA TAC ATT GAT GA-3' (SEQ ID NO:35)
[0237] This fragment was digested with Eco47111 and Xho1, and
cloned into pEGFP-1 vector in which the EGFP gene was replaced with
the rnAb179 epitope sequence. This plasmid is now referred to as
pmAb9/GFP(SR.alpha.), and was used for screening novel chimeric
promoter sequences in vitro.
[0238] Shuffling CMV Promoter Sequences and Preparation of Plasmid
Libraries
[0239] AD 169, Rhesus, Towne, and Vervet monkey CMV promoter
sequences were "shuffled" using DNA shuffling methods and
recombination formats described by the present inventors and
co-workers in co-pending applications Ser. No. PCT/US99/03022,
filed Feb. 10, 1999, PCT/US95/02126, filed Feb. 17, 1995, Ser. No.
PCT/US98/00852, filed Jan. 16, 1998, Serial No. PCT/US99/03020,
filed Feb. 10, 1999, Serial No. PCT/US99/02944, filed February 10,
1999, Ser. No. PCT/US99/03023, filed Feb. 10, 1999, Ser. No.
PCTIUS/97/24239, filed Dec. 17, 1997, U.S. Ser. No. 08/621,859,
filed Mar. 25, 1996, U.S. Ser. No. 08/621,430, filed Mar. 25, 1996,
U.S. Ser. No. 08/675,502, filed Jul. 3, 1996, Ser. No.
PCT/US96/05480, filed Apr. 18, 1996, U.S. Ser. No. 08/721,840,
filed Sep. 27, 1996, Ser. No. PCT/US97/17300, filed Sep. 26, 1997,
and U.S. Pat. No. 5,605,793, U.S. Pat. No. 5,830,721, U.S. Pat. No.
5,811,238, U.S. Pat. No. 5,837,458, U.S. Pat. No. 5,834,252; and
Stemmer, Science 270:1510 (1995); Stemmer et al., Gene 164:49-53
(1995); Stemmer, Bio/Technology 13:549-553 (1995); Stemmer, Proc.
Natl Acad. Sci. U.S.A. 91:10747-10751 (1994); Stemmer, Nature
370:389-391 (1994); Crameri et al., Nature Medicine 2(1):1-3
(1996); Crameri et al., Nature Biotechnology 14:315-319 (1996),
each of which is incorporated herein by reference in its entirety
for all purposes. DNA shuffling is also sometimes referred to as
molecular breeding directed molecular evolution (i.e., shuffling
plus screening assays), evolution, or recursive sequence
recombination.
[0240] Other methods for obtaining libraries of recombinant
polynucleotides and/or for obtaining diversity in nucleic acids
used as the substrates for shuffling include, for example,
homologous recombination (PCT/US98/05223; Publ. No. WO98/42727);
oligonucleotide-directed mutagenesis (for review see, Smith, Ann.
Rev. Genet. 19:423-462 (1985); Botstein and Shortle, Science
229:1193-1201 (1985); Carter, Biochem. J. 237:1-7 (1986); Kunkel,
"The efficiency of oligonucleotide directed mutagenesis" in Nucleic
acids & Molecular Biology, Eckstein and Lilley, eds., Springer
Verlag, Berlin (1987)). Included among these methods are
oligonucleotide-directed mutagenesis (Zoller and Smith, Nucl. Acids
Res. 10:6487-6500 (1982), Methods in Enzymol. 100:468-500 (1983),
and Methods in Enzymol. 154:329-350 (1987)) phosphothioate-modified
DNA mutagenesis (Taylor et al., Nucl. Acids Res. 13:8749-8764
(1985); Taylor et al., Nucl. Acids Res. 13:8765-8787 (1985);
Nakamaye and Eckstein, Nucl. Acids Res. 14:9679-9698 (1986); Sayers
et al., Nucl. Acids Res. 16:791-802 (1988); Sayers et al., Nucl.
Acids Res. 16:803-814 (1988)), mutagenesis using uracil-containing
templates (Kunkel, Proc. Nat'l. Acad. Sci. USA 82:488-492 (1985)
and Kunkel et al., Methods in Enzymol. 154:367-382)); mutagenesis
using gapped duplex DNA (Kramer et al., Nucl. Acids Res.
12:9441-9456 (1984); Kramer and Fritz, Methods in Enzymol.
154:350-367 (1987); Kramer et al., Nucl. Acids Res. 16:7207
(1988)); and Fritz et al., Nucl. Acids Res. 16:6987-6999 (1988)).
Additional suitable methods include point mismatch repair (Kramer
et al., Cell 38:879-887 (1984)), mutagenesis using repair-deficient
host strains (Carter et al., Nucl. Acids Res. 13:4431-4443 (1985);
Carter, Methods in Enzymol. 154:382-403 (1987)), deletion
mutagenesis (Eghtedarzadeh and Henikoff, Nucl. Acids Res. 14:5115
(1986)), restriction-selection and restriction-purification (Wells
et al., Phil. Trans. R. Soc. Lond. A 317:415-423 (1986)),
mutagenesis by total gene synthesis (Nambiar et al., Science
223:1299-1301 (1984); Sakamar and Khorana, Nucl. Acids Res.
14:6361-6372 (1988); Wells et al., Gene 34:315-323 (1985); and
Grundstrom et al., Nucl. Acids Res. 13:3305-3316 (1985). Kits for
mutagenesis are commercially available (e.g., Bio-Rad, Amersham
International, Anglian Biotechnology).
[0241] Transfection and Staining of Cells for FACS Sorting
[0242] HeLa cells were seeded at 1.times.10.sup.6 cells into 100 mm
culture dishes, and transfected with 0.5 .mu.g plasmid DNA 18-20
hours later. Transfections were performed using Superfect (Qiagen,
Valencia, Calif.) according the manufacturer's protocol. After
incubating at 37.degree. C. overnight, the cells were trypsinized,
and stained for expression of the cell surface marker using mAb179,
followed by phycoerythrin (PE)-labeled goat anti-mouse
immunoglobulin (Ig) (Caltag; Burlingame, Calif.). Cells were sorted
using a FACStar, or FACSVantage (Becton Dickinson; San Jose,
Calif.) to collect those that expressed high levels of the mnAb179
epitope and relatively low levels of EGFP. The staining
concentration was determined for each labeled protein to provide a
maximal Mean Fluorescence Intensity (NFI) and minimal background
signal (e.g., optimum staining concentration was the concentration
per 10.sup.6 cells). For a detailed description of flow cytometry
cell sorting methods and staining methods, which are known in the
art, see Current Protocols in Immunology, John Colligan et al.,
eds., Vols. I-IV (John Wiley & Sons, Inc., 2001 Supplement) and
Rapley, R. and Walker, J. M. eds., Molecular Biomethods Handbook
(Humana Press, Inc. 1998) [hereinafter "Rapley and Walker"], each
of which is incorporated herein by reference in its entirety for
all purposes.
[0243] HIRT Extraction of Plasmids
[0244] Plasmids were recovered from the sorted cells by Hirt
preparation as follows. The sorted cells were pelletted by
centrifugation, and resuspended in 125 microliter (.mu.L) phosphate
buffered saline (PBS). An equal volume of 2.times. HIRT buffer
(1.2% sodium dodecyl sulfate (SDS), 20 milliMolar (mM) EDTA pH 8.0)
was added to the cells and the cell samples incubated at room
temperature for 15 minutes to allow the cells to lyse. After the
addition of 62 .mu.L 5 Molar (M) NaCl to give a final concentration
of 1 M, the samples were placed at 4.degree. C. overnight. The
samples were then centrifuged at 14,000.times. g for 60 minutes
(min) at 4.degree. C., and the supernatant extracted with an equal
volume of phenol-chloroform. The DNA was precipitated with cold
ethanol, and washed with room temp 70% ethanol. Finally, the pellet
was air dried, and the DNA resuspended in 10 mM Tris-HCl pH
7.4.
[0245] Preparing an "Enriched" Plasmid Library
[0246] Enriched plasmid libraries were prepared by transformation
of XL-10 ultracompetent cells with DNA extracted by the HIRT
method. Transformed cells were plated on agarose plates containing
40 .mu.g/mL (40 micrograms/milliliter)Kanamycin, and incubated at
37.degree. C. overnight. The resulting colonies were scraped,
washed in LB, and plasmid DNA prepared using Qiagen's
Endotoxin-free Maxiprep kits (Qiagen; Valencia, Calif.).
[0247] Plasmid Preparation in 96-well Format
[0248] Plasmid libraries were transformed into E. coli XL-10
ultracompetent cells, and spread on agar plates containing
Kanamycin. Individual colonies were picked into 1.2 mL Terrific
broth supplemented with Kanamycin in 96-well blocks. The block
cultures were incubated for 20 hours at 37.degree. C. with shaking.
Bacteria were pelleted by centrifugation, and plasmids prepared
robotically in a 96-well format. DNA yields were determined by
reading optical densities (ODs) at 260 and 280 nanometer (nm) on a
SpectraMax plate reader (Molecular Devices; Sunnyvale, Calif.). DNA
concentrations typically varied between 100 and 200 ng/.mu.L.
[0249] 96-well Format Transfections of Mammalian Cells
[0250] HeLa cells were maintained in DMEM (Gibco; Grand Island,
N.Y.) with 10% FCS (Hyclone; Logan, Utah), and
Penicillin/Streptamycin. They were seeded at 2.times.10.sup.4
cells/well into 96-well plates, and transfected with 0.5-1 .mu.g
(micrograms) DNA 18 hours later using Qiagen's Superfect, according
to the manufacturer's protocol. The cells were incubated at
37.degree. C. for 20-24 hours, and stained for FACS analysis using
mAbl79 and PE-labelled goat anti-mouse Ig (Caltag; Burlingame,
Calif.). Analysis was performed using a FACScan or FACSCalibur with
CellQuest software (Becton Dickinson; San Jose, Calif.).
[0251] Construction of Vectors for Testing Wild-type CMV Promoters
and Novel Chimeric Promoter Sequences in vivo
[0252] The .beta.-galactosidase gene was amplified by PCR from
plasmid pCMV.beta. using the following Nhe1- and Apa1-encoding
primers:
6 5'-AAG CTG GCT AGC ATG TCG TTT ACT TTG ACC AAC-3'(SEQ ID NO:36)
5'-AAA CGG GCC CTT ATT TTT GAC ACC AGA CCA AC-3'(SEQ ID NO:37)
[0253] The resulting fragment was digested with Apa1 and Nhe1 and
cloned into plasmid pcDNA3.1.
[0254] Preparation of Plasmids for Injection into Mice
[0255] Plasmids for injection were prepared using Qiagen Endofree
Maxiprep DNA kits (Qiagen; Valencia, Calif.), and resuspended in
PBS at 0.1 or 0.2 mg/mL for injection. Each preparation was assayed
for endotoxin using a Limulus Amebocyte Lysate assay kit
(Biowhittaker; Walkersville, Md.), and contained less than 60
EU/.cndot. g (enzyme units/microgram) plasmid DNA.
[0256] Injection of Mice with Plasmid DNA
[0257] Mice were injected in the tibialis anterior (TA) muscle with
a volume of 50 .mu.L plasmid in PBS.
[0258] Collection and Preparation of Samples from Mice
[0259] Blood was collected from the lateral tail vein of mice, and
serum harvested following centrifugation. Sera samples were stored
at -20.degree. C. until required for ELISA (Enzyme Linked
Immunosorbent Assay). Individual TA muscles were excised,
homogenized in 0.5 mL of Promega Cell Culture Lysis Reagent
(Madison, Wis.), and the homogenates stored at -20.degree. C.
Samples were thawed, centrifuged at 1400.times. g at 4.degree. C.,
and the supernatants collected to assay for Luciferase and protein
content.
[0260] Injection of Human Fetal Muscle with Plasmid DNA
[0261] Human fetal limbs were obtained from (Advanced Biosciences
Resources Inc.) for testing the activities of promoter sequences in
human muscle. Plasmid DNA was diluted to 225 .mu.g/300 .mu.L of PBS
and three aliquots of 100 .mu.L each were injected into TA muscle.
Muscle tissue was harvested after 48 hours, homogenized and assayed
for Luciferase content using the Promega Luciferase Reporter Assay
System described herein and as set forth in Promega Technical
Bulletin No. 101 entitled "Luciferase Assay System" [hereinafter
Promega Tech Bulletin No. 101], which is incorporated herein by
reference in its entirety for all purposes.
[0262] Assay for Luciferase Gene Expression
[0263] The firefly luciferase gene is highly effective as a genetic
reporter gene for measuring gene expression. The luciferase assay
yields luminescence through an ATP-dependent oxidation of
luciferin. Light intensity is a measure of the rate of catalysis by
luciferase. Luciferase enzyme activity of the muscle tissue extract
was measured on a microplate luminometer (or scintillation counter)
using the Luciferase Reporter 1000 Assay System from Promega
(Madison, Wis.), according to the manufacturer's instructions, as
set forth in Promega Tech Bulletin No. 101. Luciferase enzyme assay
methods described in Manthorpe, M. et al., Human Gene Therapy
4:419-431 (1993) [hereinafter Manthorpe et al.], which is
incorporated herein by reference in its entirety for all purposes,
can also be employed.
[0264] Measurement of Antibodies to .beta.-galactosidase by
ELISA
[0265] .beta.-galactosidase (".beta.gal") antibody-containing serum
samples were diluted in 96-well plates which had been coated with 4
.mu.g/mL .beta.gal (Sigma; St Louis, Mo.). Antibody binding (bound
antibodies) was detected using peroxidase-conjugated anti-mouse IgG
immunoglobulin (1/5000 dilution Sigma; St Louis, MO) followed by 3,
3', 5, 5' tetramethyl benzidine (TMB) substrate (Pierce; Rockford,
Ill.). The reaction was stopped by the addition of 2 Normal (N)
H.sub.2SO.sub.4, and the absorbance read at 450 nm on a SpectraMax
plate reader (Molecular Dynamics; Sunnyvale, Calif.). Endpoint
antibody titers were defined as the reciprocal of the highest
dilution of serum giving detectable signal 3 standard deviations
above background. FIG. 5 shows the results of reciprocal endpoint
Ab titers (+/- SEM) for selected shuffled clone and parental
clones. For a description of the ELISA assay screening method for
anti-.beta.-galactosidase antibodies used herein, which is known in
the art, see Current Protocols in Immunology, John Colligan et al.,
eds., Vols. I-IV (John Wiley & Sons, Inc., 2001 Supplement),
and Forg, P., Gene Therapy 5:7890797 (1998), each of which is
incorporated herein by reference in its entirety for all purposes.
As a control, uninjected mice were used. The vector control
comprised a promoterless plasmid encoding .beta.-galactosidase
injected into mice in similar manner.
[0266] Results
[0267] Generation of a Library of Novel Chimeric Promoter
Sequences
[0268] A library of chimeric promoter/enhancer sequences was
created by family DNA shuffling of wild-type sequences from four
related strains of CMV. The promoter and enhancer sequences were
obtained by PCR from the AD169 and Towne human CMV strains.
Similarly, the promoter and enhancer sequences were obtained from
rhesus and vervet monkey CMVs by amplification. The
promoter/enhancer nucleic acid sequences of the two human CMV
strains are 97.5% identical, and share 50-70% identity with the
nucleic acid sequences of the two monkey isolates, depending on the
region of the sequence analyzed. (For example, the homology of
these sequences was higher in the region of the transcription start
site; see FIGS. 8 and 10.) The sequences taken together are
referred to herein as "promoters."
[0269] The shuffled nucleotide sequences from the shuffled
nucleotide library were cloned into plasmid pmAb179/GFP(SR.alpha.)
and used to direct transcription of a marker gene (mAb179 epitope)
in mammalian cells. The plasmid expression vector also encodes an
internal marker (EGFP) under the control of the SR.alpha. promoter.
This internal marker under the control of this promoter allows for
analysis and sorting of cells harboring equal numbers of
vectors.
[0270] Other expression markers (such as luciferase,
.beta.-galactosidase, lacZ, and green fluorescent protein) can also
be used in this type of assay.
[0271] In vitro Screening of Libraries Comprising Novel Chimeric
Promoter Sequences Resulting from Shuffling of CMV Promoter
Sequences
[0272] A tiered screening process was applied to the library to
identify those shuffled (chimeric) sequences that gave the highest
levels of reporter gene expression (FIG. 1). First, the plasmid
library was enriched for good promoter sequences by transfection
and FACS sorting those cells expressing the highest levels of
marker gene, relative to expression of the internal marker to
account for differences in plasmid vector copy numbers per cell.
Plasmids were extracted from the sorted cells by HIRT preparation
to generate "enriched libraries."
[0273] The increase in frequency of clones directing higher levels
of transgene expression after just one round of FACS sorting is
demonstrated in FIG. 2. Individual clones from the round 1 shuffled
chimeric promoter library and the enriched library were included in
plasmid vectors, the plasmid vectors introduced into mice, and
mouse cells were subsequently assayed by FACS analysis. A plasmid
comprising a shuffled nucleic acid sequence for each clone was
introduced into mice. A plasmid comprising a wild-type (WT) human
CMV promoter Towne strain nucleic acid sequence was introduced into
6 mice for comparison with the original library analysis of
selected clones; a plasmid comprising a WT human CMV promoter Towne
strain nucleic acid sequence was introduced into 8 mice for
comparison with the enriched library analysis of selected clones.
For each analysis, the mean value for the WT transfections is shown
in FIG. 2 by the arrow in the graph. FIG. 2 shows the distribution
of expression levels, as measured by flow cytometry, of
individually analyzed CMV promoter clones in the original library
versus the enriched FACS-sorted library. Cells were sorted using a
FACStar or FACSVantage to collect those cells with clones that
expressed high levels of the mAb179 epitope and relatively low
levels of EGFP. Reporter gene expression was measured by the Mean
Fluorescence Intensity (MFI) by standard FACS sorting methods. As
shown in FIG. 2, the FACS-sorted library enriched the population
for high-activity promoters. A higher frequency of strongly
expressing clones was observed in the enriched library. In each
graph in FIG. 2, a relatively high signal was shown for clones
having no or little reporter gene expression; this signal likely
corresponds to cells transfected with plasmids comprising dead or
inactive promoters, cells transfected with plasmids lacking a
promoter (i.e., control vector), and untransfected cells.
[0274] Plasmid DNA was then prepared robotically from individual
clones (picked from the enriched libraries) for transfection of
cells in 96-well trays. Cells were transfected with a plasmid DNA
comprising a shuffled promoter nucleic acid sequence, a plasmid DNA
comprising a wild-type parental promoter sequence, or a DNA vector
lacking a promoter (which served as the vector control).
Transfected cells were screened by FACS to determine the level of
expression of the cells of the reporter gene (maker gene), relative
to the internal marker. FACS screening identified those cells that
expressed the highest levels of marker gene, relative to the
internal marker. The results are shown in FIG. 3. The individual
clone identification (Clone ID) names are shown along the X-axis
(FIG. 3). Results for vector control and parental clones are
presented in lightly shaded bars; dark bars represent shuffled
clones. Results are expressed as mean.+-.SD (standard deviation)
for 4 independent transfections. For each chimeric promoter clone,
the level of expression of the reporter gene is shown. These assays
revealed the diversity of promoter activities generated by DNA
shuffling.
[0275] Two rounds of shuffling, enrichment by FACS sorting, and
screening of individual clones in vitro were completed. Following
enrichment of the first round library by FACS sorting, 1000
individual clones were screened by transfection and FACS analysis;
the best 18 clones from these assays were chosen as starting
sequences for generating a second round library. This library was
enriched by two successive rounds of FACS sorting before 1000
individual clones were screened in transfection and FACS
assays.
[0276] In vivo Screening ff Libraries Comprising Novel Chimeric
Promoter Sequences Resulting from Shuffling of CMV Promoters
[0277] Thirty of the chimeric promoter sequences that produced the
highest levels of expression of the reporter genes in the in vitro
analyses were subcloned into DNA vaccine vectors encoding a
reporter molecule (i.e., Luciferase or .beta.-galactosidase) for in
vivo studies of gene expression and immune response. The chimeric
promoter sequences were positioned to drive expression of the
respective reporter genes. Each chimeric promoter sequence was
operably linked to a Luciferase or .beta.-galactosidase gene.
[0278] Individual plasmid preparations comprising a promoter
sequence operably linked to a reporter gene were inoculated
intramuscularly (via the tibialis anterior (TA) muscle) into groups
of 5 to 10 mice for each clone. Plasmids comprising a parental
sequence operably linked to the luciferase reporter gene were also
injected into groups of mice in a similar manner (for each of the
four parental sequences) and used for comparison with the plasmids
comprising chimeric promoter sequences. As a vector control, an
empty vector including the luciferase reporter gene, but lacking a
promoter, was injected into mice in a similar manner. A group of
mice that were not inoculated with any vector served as a control
group ("Control"). Expression of luciferase in homogenates of the
TA muscle and serum antibody titers against .beta.-galactosidase
were then measured as an indication of promoter activity. From
these results, 5 luciferase clones and 6.beta.-galactosidase clones
were chosen for further studies to confirm the activities of the
promoter sequences in vivo.
[0279] 1. In Vivo Screening Assay to Detect Luciferase Gene
Expression
[0280] The amount of Luciferase expression in TA muscles of mice
was determined at various time point(s) after injection. In the
present example, the amount Luciferase expression in TA muscles was
measured 7 days after injection of 10.cndot.g plasmid per muscle
(FIG. 4). The linear range of light production was determined
according to the manufacturer's instructions (Promega Tech Bulletin
No. 101). Cell extracts were prepared and assays were performed
according to the manufacturer's instructions (Promega Tech Bulletin
No. 101). Light production by luciferase (luciferase activity) was
measured according to the manufacturer's instructions (Promega Tech
Bulletin No. 101) by relative light units (light intensity) using a
luminometer or scintillation counter (reflected as counts per
minute (cpm) (+/-SEM) (standard error of the mean)). See also
Manthorpe et al., supra. Results are shown in FIGS. 4 and 5. In
FIG. 4, results are expressed as mean.+-.SEM for 32 samples.
[0281] The transgene (reporter) expression by shuffled promoters
was statistically significantly higher in selected clones than that
induced by one or more of the four parental wild-type promoters.
Shuffled clone 6A8 was found to give the highest levels of
Luciferase expression of the chimeric promoter sequences tested,
and performed approximately 2-fold better than the best parental
sequences, human AD169 and Towne (p<0.05, t-test), as is shown
in FIG. 5. Results are expressed as mean.+-.SEM for 32 samples.
Clones 6D4 and 6F6 yielded levels of luciferase similar to that
observed with the parental sequences, with clones 9G7 and 9G12
giving lower levels, comparable to the Rhesus and Vervet parental
sequences (FIG. 4). Luciferase expression from a promoterless
luciferase-encoding plasmid vector (pcDNALuc) was negligible. The
control mice (non-injected) also showed no measurable expression
levels.
[0282] 2. In Vivo ELISA Screening Assay for
Anti-.beta.-Galactosidase Antibodies
[0283] Mice were injected with 10 .mu.g
.beta.-galactosidase-encoding plasmids on days 0 and 15, and serum
collected on days 14 and 28 for measurement of
anti-.beta.-galactosidase antibodies. Plasmids comprising a AD 169,
Towne, or Vervet parental nucleic acid sequence operably linked to
.beta.-galactosidase nucleic acid sequence were also injected into
groups of mice in a similar manner and used for comparison with the
plasmids comprising the chimeric promoter sequences. As a vector
control, an empty vector comprising a promoterless
.beta.-galactosidase-encoding plasmid (pcDNA.beta.-gal) was
injected into mice in a similar manner. A group of mice that were
not inoculated with any vector served as a control group. FIG. 6A
and 6B shows the antibody titer levels measured in serum by ELISA
methods, where the serum was obtained following injection of mice
with .beta.-galactosidase-encoding plasmids (10 .mu.g or 4 .mu.g
plasmid, respectively) at the time (day) noted above.
[0284] Injection of the shuffled clone, 11E2, gave the strongest
antibody response against .beta.-galactosidase at day 14
post-injection, while clone 6B2 gave the strongest response at day
28 post-injection. Results are expressed as mean.+-.SEM for 8-20
samples.
[0285] Antibody titers in mice injected with clone 6B2 were
approximately 2-fold higher than in those injected with clones
carrying the (best) wild-type parental promoters. Clone 6B2
displayed about a 2-fold higher transgene expression in vivo than
the parental promoters. All other chimeric clones tested gave
comparable antibody titers at day 28 to the parental clones. Mice
injected with promoterless .beta.-galactosidase-enc- oding plasmid
gave a negligible antibody response. The control group of mice
(uninjected) also showed negligible antibody response.
[0286] Assessment of Novel Chimeric Promoterfunction in Human
Muscle
[0287] The expression of Luciferase in human fetal muscle tissue
was assessed following injection into such tissue of a DNA plasmid
comprising a luciferase gene and the nucleic acid sequence
corresponding to clone 6A8 or parental human clone AD169 or Towne.
A similar plasmid vector, but lacking a promoter, was injected in a
similar manner as a control vector. Luciferase levels in samples of
the homogenate of human fetal muscle prepared 2 days after
injection of luciferase-encoding plasmids were measured; these
levels were found to be comparable and significantly higher than
observed in samples from muscles injected with the promoterless
vector (FIG. 7). Results are expressed as mean +SEM for 3-6
injections for each clone.FIG. 7 confirms that the chimeric
promoter 6A8 was functional in human muscle tissue.
[0288] Analysis of Chimeric Promoter DNA Sequences for High-level
Expression
[0289] Sequence analysis of selected shuffled chimeric promoters
revealed that they comprised mainly nucleic acid sequences from the
AD169 and Towne human parental nucleic acid sequences. In addition,
the sequences contain between 2 and 17 unique nucleotides
throughout the promoter. Deletions of one or two nucleotides occur
in several of the clones, and 11E2 also has an additional
nucleotide (nt) after nt853 (numbering is based on the consensus
sequence as shown in FIG. 8). Clones 6F6, 9G7, 11E2, and 12C9
contain nucleotide sequences derived from the Rhesus monkey exon A
approximately from nt817 (which is close to the transcription start
site) to nt863. Clones 4B5, 6B2, 6D4, and 12E1 have a deletion
corresponding to the region 684-735 nucleotides in the consensus
sequence. Clone 12C9 is truncated at nucleotide (nt) residue 909
(numbered according to the consensus sequence shown in FIG. 8).
Notably, clone 12C9 gave a comparable or increased antibody
response in the B-gal screening assay relative to other chimeric
clones or the parental sequences despite having a truncated
sequence. Compared with the human AD169 and Towne nucleic acid
sequences, the 12C9 nucleic acid sequence lacks a short segment of
the nucleic acid sequence corresponding to the first exon and
intron of each of the AD169 and Towne strains.
[0290] There is also a deletion in clone 9E1 corresponding to
nucleotides 319 to 512 in the parental clones. In all of the
shuffled sequences, the TATA box (or TATATAA box), CAAT (or CAAAT
box) box and transcription start site (T=thymine, C=cytosine,
A=adenine nucleotide bases) are identical to those found in the
AD169 and Towne parental sequences (see FIG. 8). For known CMV
promoters, it is generally believed the TATA box is important for
promoter activity.
[0291] Several of these mutations occur in regions of repeated
elements that occur in the CMV enhancer and are rich in
transcription factor binding sites. Most notable is the deletion in
clone 9E1 from nucleotides 319 to 512, which eliminates a whole 21
bp repeat element, and parts of two others, three 19 bp repeat
elements, and one each of the 18 and 16 bp repeat elements. This
likely accounts for the low expression of the mAb179 epitope
reporter gene when cells were transfected with clone 9E1.
[0292] Screening of Chimeric Promoter DNA Sequences for Low- or
Intermediate-level Transgene Expression
[0293] A library of chimeric promoter sequences with diverse
activities by DNA shuffling of CMV promoters sequences from four
related strains of CMV promoter (two human strains, Towne and AD
169; and Vervet and Rhesus monkey strains) were generated using
methods described above. For example, the major IE region
promoter/enhancer regions of the resulting library of chimeric
promoter nucleic acids was screened to identify those chimeric
variants that gave a level expression of reporter genes in vitro
lower than the reporter gene expression level produced by one of
the parental genes, using the procedures outlined above.
[0294] Those chimeric promoter clones identified as directing lower
levels of reporter gene expression in vitro were individually
isolated, cloned into plasmid vectors, and transfected in vivo into
mammalian cells. The cells were screened to identify those chimeric
variants that gave high-level expression of reporter genes in
vivo.
[0295] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. It is understood that the
examples and embodiments described herein are for illustrative
purposes only and that various modifications or changes in light
thereof will be suggested to persons skilled in the art and are to
be included within the spirit and purview of this application and
scope of the appended claims. For example, all the techniques and
apparatus described above may be used in various combinations. All
publications, patents, patent applications, and/or other documents
cited in this application are incorporated herein by reference in
their entirety for all purposes to the same extent as if each
individual publication, patent, patent application, and/or other
document were individually indicated to be incorporated herein by
reference in its entirety for all purposes.
* * * * *
References